Alpha-Amylase Variants Stabilized Against Dimerization and/or Multimerization, Method for the Production Thereof, and Detergents and Cleansers Containing these Alpha-Amylase Variants

ABSTRACT

The present invention relates to α-amylase variants that are stabilized to dimerization and/or multimerization, in particular at elevated temperatures or high pH, by point mutagenesis of positively polarized or charged or neutral surface amino acids to give more negatively polarized or charged amino acids. The invention further relates to methods of increasing the stability of an α-amylase to dimerization and/or multimerization brought about by electrostatic interactions whereby at least one amino acid residue on the surface of the starting molecule, which makes a neutral or positively polar or charged contribution to the electrostatic potential of said molecule, is replaced with a more negatively polar or negatively charged amino acid residue. The α-amylase variants obtained thereby exhibit better stability to influences of the solvent, increased processivity, and are suited for numerous industrial areas of use, in particular as active ingredients in detergents and cleansers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/692,691, filed Mar. 28, 2007, the entire contents of which are herebyincorporated by reference herein. U.S. application Ser. No. 11/692,691is a continuation application of PCT/EP2005/010259 filed Sep. 22, 2005,which claims the priority of German patent application DE 10 2004 047776.0, filed Oct. 1, 2004. Each of the foregoing applications isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to α-amylase variants which have beenstabilized to a di- and/or multimerization brought about byelectrostatic interactions, by means of point mutagenesis of surfaceamino acids, to methods of preparation thereof via mutagenesis of aminoacid residues with a contribution to the electrostatic potential of themolecule and to detergents and cleansers containing these α-amylasevariants.

BACKGROUND OF THE INVENTION

α-Amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds ofstarch and starch-like polymers with the formation of dextrins andβ-1,6-branched oligosaccharides. They are very much among theindustrially most important enzymes. Thus, for example, α-amylases areemployed in the production of glucose syrup, for the treatment of rawmaterials in the manufacture of textiles, for the production ofadhesives or for the production of sugar-containing food or foodingredients. Another important field of use is the use as an activecomponent in detergents and cleansers.

Since detergents and cleansers have mainly alkaline pH values,particular use is made here of α-amylases that are active in alkalinemedium. These types of α-amylases are produced and secreted bymicroorganisms, i.e. fungi or bacteria, especially those of the generaAspergillus and Bacillus. Starting from these natural enzymes, there isnow quite a vast abundance of variants available which have been derivedvia mutagenesis and have specific advantages depending on the field ofuse.

Examples thereof are the α-amylases of Bacillus licheniformis, B.amyloliquefaciens and B. stearothermophilus and their improveddevelopments for the use in detergents and cleansers. The B.licheniformis enzyme is available from Novozymes under the nameTermamyl® and from genencor under the name Purastar® ST. Products offurther development of this α-amylase can be obtained from Novozymesunder the trade names Duramyl® and Termamyl® ultra, from genencor underthe name Purastar® OxAm and from Daiwa Seiko Inc., Tokyo, Japan, asKeistase®. The B. amyloliquefaciens α-amylase is sold by Novozymes underthe name BAN®, and variants derived from the B. stearothermophilusα-amylase are sold under the names BSG® and Novamyl®, likewise byNovozymes.

Examples of α-amylases from other organisms are further developments ofthe Aspergillus niger and A. oryzae α-amylases, obtainable under thetrade name Fungamyl® from Novozymes. Another commercial product isAmylase-LT®, for example.

Mention may further be made of the Bacillus sp. A 7-7 (DSM 12368)α-amylase disclosed in the application WO 02/10356 A2 and of the B.agaradherens (DSM 9948) cyclodextrin glucanotransferase (CGTase)described in the application WO 02/44350 A2. In addition, for example,the applications WO 03/002711 A2 and WO 03/054177 A2 define sequencespaces of α-amylases, all of which could be suitable in principle forcorresponding applications.

The application DE 10309803.8, which has not been prepublished, forexample, describes point mutations for improving the activity of saidenzymes in alkaline medium. According to this application, amino acidsubstitutions suitable here are those in positions 13, 32, 194, 203,230, 297, 356, 406, 414 and/or 474, according to the numbering of theunprocessed B. amyloliquefaciens α-amylase.—These positions areaccording to the numbering of the unprocessed Bacillus sp. A 7-7 (DSM12368) α-amylase (WO 02/10356 A2) L13, T36, W198, S201, I208, A235,D302, D361, H408, K416 and N476, respectively, with the following,particularly effective substitutions: 13P, 32A, 194R, 197P, 203L, 230V,297D, 356D, 406R, 414S and 474Q.

Another example of point mutagenesis on α-amylases is the application WO00/22103 A1 which discloses polypeptides, inter alia also α-amylasevariants, containing mutagenized surface amino acids. The purpose ofthis mutagenesis was to reduce the immunogenicity and/or allergenicitycaused by these molecules.

Fusion products of α-amylases for the use in detergents and cleansershave also been described. Thus, for example, the application WO 96/23874A1 discloses hybrids of the α-amylases of Bacillus licheniformis, B.amyloliquefaciens and B. stearothermophilus. According to the teachingof this application, such hybrid amylases may be prepared fordetermining the three-dimensional structure of said amylases, in orderto use said structure for detecting important positions for enzymicactivity. Further developments in this respect are the applications WO97/41213 A1 and WO 00/60059 A2, which report numerous α-amylase variantswhose respective performances have been improved. The application WO03/014358 A2 discloses special hybrid amylases of B. licheniformis andB. amyloliquefaciens.

The three applications WO 96/23873 A1, WO 00/60060 A2 and WO 01/66712A2, which are the basis of the commercial product Stainzyme® fromNovozymes, constitute another important prior art. All the variantsobtainable by point mutagenesis which are specified in each of theseapplications have altered enzymic properties and are therefore claimedor described for the use in detergents and cleansers. WO 96/23873 A1makes mention of, in some cases two or more, point mutations in morethan 30 different positions in four different wild type amylases. Theyapparently have altered enzymic properties with regard to thermalstability, oxidation stability and calcium dependence. They includepoint mutations in the following positions, each of which is stated withrespect to the Bacillus sp. NCIB 12512 α-amylase: substitution ofoxidizable amino acids in positions M9, M10, M105, M202, M208, M261,M309, M382, M430 or M440, preferably M91L; M10L; M105L; M202L,T,F,I,V;M208L; M261 L; M309L; M382L; M430L and M440L; deletions of F180, R181,G182, T183, G184 and/or K185; and additionally the substitutions K269R;P260E; R124P; M105F,I,L,V; M208F,W,Y; L2171; V2061,L,F; Y243F; K108R;K179R; K239R; K242R; K269R; D163N; D188N; D192N; D199N; D205N; D207N;D209N; E190Q; E194Q or N106D.

The application WO 00/60060 A2 likewise specifies a multiplicity ofpossible amino acid substitutions in 10 different positions, on thebasis of two very similar α-amylases from two different microorganisms,with the same numbering α-amylases AA349 and AA4560). These are thefollowing sequence variations: R181*, G182*, D183*, G184*;N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;1206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; and/orR181A,N,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V. This development too isagainst the background of improving performance via mutations.

WO 01/66712 A2, finally, refers to 31 different amino acid positionswhich are partially identical to the ones mentioned above and which havebeen mutated in either of the two α-amylases specified in theapplication WO 00/60060 A2 and which are said to improve aspects of bothperformance and stability. These are point mutations in the followingpositions: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195,M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396,R400, W439, R444, N445, K446, Q449, R458, N471 and N484, again asdefined via α-amylase AA560, i.e. also according to the numbering of theletter. Among these, the following variants are said to be particularlyadvantageous: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N,K;S94K; R118K; N125A,R,K; N174D; R181Q,E,K; G186R; W189R,K; N195F;M202L,T; Y298H,F; N299A; K302R, S303Q, N306G,D,R,K; R310A,K,Q,E,H,D,N;N314D; R320K; H324K; E345R,D,K,N; Y396F; R400T,K; W439R; R444K; N445K,Q;K446N; Q449E; R458K; N471E and N484Q.

This latter application further describes polypeptide crystals, inparticular those of enzymes, that might be improved with respect totheir resolution capacity in that, in the molecules located next to theactual molecules of interest in the crystal in question and interactingtherewith, those amino acids which are located within a distance of 6.0Å to the polypeptide of interest could be mutated. This would apply inparticular to Termamyl-like enzymes located next to other or identicalTermamyl-like enzymes in a crystal; here in particular to a distance ofless than 3.5 Å. This would concern positions 19, 20, 21, 22, 25, 28,29, 53, 76, 84, 87, 90, 93, 94, 124, 125, 126, 128, 142, 144, 156, 157,158, 159, 160, 161, 170, 171, 172, 173, 174, 175, 183, 184, 185, 186,187, 188, 189, 190, 193, 195, 196, 197, 209, 212, 226, 229, 256, 257,258, 259, 280, 281, 298, 299, 300, 302, 303, 304, 305, 306, 310, 311,314, 319, 320, 321, 322, 341, 345, 405, 406, 408, 444, 447, 448, 449,463, 464, 465, 466 and 467; and positions 22, 25, 28, 76, 94, 125, 128,158, 160, 171, 173, 174, 184, 189, 209, 226, 229, 298, 299, 302, 306,310, 314, 320, 345, 405, 447 and 466 for the shorter distance.

All of these applications regarding point mutagenesis share the factthat the point of stability is also evaluated under the aspect of a goodperformance in the corresponding field of use. This is because thestability maintained during storage and usage of α-amylases, for examplewithin the context of detergent formulations, results in a highperformance or in a performance as constantly high as possible withcorresponding usage. An increase in stability, in particular toaggregate formation, above all in the course of workup, is notdescribed.

The purpose of increasing stability is pursued by numerous applicationsdescribing special enzyme stabilizers. These additional ingredientscause a protein and/or enzyme present in corresponding agents to beprotected from damage such as, for example, inactivation, denaturationor decay, particularly during storage. Thus, reversible proteaseinhibitors form a group of stabilizers. Others, for example polyols,stabilize to physical influences such as freezing, for example. Otherpolymeric compounds such as acrylic polymers and/or polyamides stabilizethe enzyme preparation inter alia to pH fluctuations. Reducing agentsand antioxidants increase stability of the enzymes to oxidative decay.

Compounds of these kind are added to the enzymes both during applicationand in the course of their work-up, which is particularly important, ifa previously present stabilizer has been removed together with the othercontaminations in a component step of said workup, for example aprecipitation.

The prior art regarding the improvement in stability of α-amylases canbe summarized as follows: a multiplicity of α-amylase variants have beendeveloped via point mutagenesis, with the aim of these developmentshaving mainly been that of improving the performance of said α-amylasevariants. This category also includes those variants which have beenstabilized with regard to denaturing agents such as bleaches orsurfactants, since in these cases, the desired performance of the enzymeis optimized. In other cases, additional compounds which are overallreferred to as stabilizers are mainly used for increasing stability ormaintaining the physicochemical conformation.

A previously less regarded aspect in enzyme development is that ofstabilizing the molecules per se in such a way that they have increasedstability over the wild type molecule even during their workup. Anadditional advantageous effect thereof would be that this increasedstability should also benefit the intended later usage of the enzyme inquestion.

The necessity for this is particularly evident in the case ofα-amylases. At least some of these tend, especially during productionand workup, to form multimers, specifically in the form of amorphousaggregates, which precipitate irreversibly. As a result, the activitiesin question are lost even during workup. The work-up process includesall steps of industrial production, starting from isolating the enzymein question, in particular the fermentation media common inbiotechnological production, via the following washing and separationsteps (for example by precipitation) and concentration up toformulation, for example granulation. In particular, those substeps inwhich the enzyme is present in solutions with comparatively highconcentrations are critical here, because, seen statistically, morefrequent contacts occur here between the molecules than at lowerconcentrations. However, the aggregate formation may also occur duringstorage of α-amylase-containing agents or during application, forexample when used as active ingredient in washing or cleaning processes.

This problem can go as far as individual α-amylases, although they canbe produced and studied on a laboratory scale, refusing to be producedon an industrial scale with the aid of generally common methods. This isthen also referred to as said enzymes having low process stability,meaning a large variety of possible processings and uses. For example,Bacillus sp. A 7-7 (DSM 12368) α-amylase exhibits a greater tendencytoward multimerization than the native B. licheniformis α-amylase.Approaches to eliminate this type of instability, that is to say toreduce the tendency toward multimerization, would only enable suchenzymes in the first place to be accessible to production on anindustrial scale and thus to the large variety of fields of use inindustrially relevant quantities.

SUMMARY OF THE INVENTION

It was therefore the object to stabilize α-amylases, in particular thatof Bacillus sp. A 7-7 (DSM 12368), per se in such a way that they have,compared to the starting molecule, increased stability to aggregateformation.

In one partial aspect of said object, first a possible cause of theirtendency toward aggregate formation, which is based on the structure ofthe enzymes in question, had to be determined. Subsequently, said causewas to be answered by suitable structural modifications.

Said structural modifications would firstly have an advantageous effecton the workup, i.e. industrial production, of said enzymes. Secondly,they should also be advantageous for the use of α-amylases, for examplein detergents and cleansers, because this should additionally beaccompanied by a constantly high activity during said use.

Particularly advantageous solutions to this object would therefore beconsidered to be those α-amylases which, besides said stability toaggregate formation, exhibit further positive properties with regard totheir intended use, in particular with regard to their use in detergentsand cleansers.

This applied especially to Bacillus sp. A 7-7 (DSM 12368) α-amylase.Preference is nonetheless given to those solutions which can betransferred to other α-amylases.

On the way to solve said object, the presence of areas with differentelectrostatic potential on the surface of α-amylases in their native,correctly folded globular structure was contemplated as a reason for thephenomenon of aggregate formation, which phenomenon is observed inparticular with α-amylases, in particular with certain α-amylases. Thus,for example, large surface areas of Bacillus sp. A 7-7 (DSM 12368)α-amylase are negatively charged, while other, sometimes large areashave a positive or neutral electrostatic potential. Thus it is possiblefor a plurality of molecules, due to electrostatic interactions betweenthe differently polarized or charged areas thereof, to assemble andthereby result in dimerization up to the formation of multimers. Thisinteraction can be removed by the addition of denaturing agentspresumably only with impairment of the globular structure, i.e. with therisk of irreversible inactivation. Secondly, said interaction per se mayresult in irreversibly inactive aggregates, in particular if the mutualattractor forces are strong enough in order to influence the molecularstructure, even without intervention from the outside.

A molecular-biological approach is pursued in order to solve the objectin question. The reason is that this influences the enzymic propertiesmediated by the mere amino acid sequence. In this connection, it wassurprisingly found that modifications, i.e. point mutations which makethe charge pattern on the surface of these molecules more similar to oneanother, counteract aggregate formation.

The amino acid residues present “on the surface of these molecules” canbe defined by the 3D structure of the globular enzyme protein, the“Conolly surface” or the “accessibility” value, i.e. solventaccessibility (see below). The observed positive, negative or neutralcontribution to the electrostatic potential of the molecule results fromthe chemical properties of the particular amino acid residues under theinfluence of the in each case next amino acid residues, in particularthose below the surface, and may be calculated as illustrated below. Theobserved aggregation appears to occur in particular if a plurality ofthe residues immediately adjacent on the surface have the same pluralityor charge.

Without wishing to be bound to this theory, it can be assumed that theformation of α-amylase aggregates, at least a significant part thereof,may be attributed to a di- and/or multimerization via differently polarand/or different charged surface areas of these protein molecules. As aresult thereof, said molecules tend to align themselves in a regularorientation, similar to magnets. Breaking the two-dimensionality of thepositively polarized or charged areas in favor of the predominantnegative charge thus stabilizes the α-amylases to aggregate formationbased on multimerization. This is beneficial both to isolation (forexample in a precipitation step) and storage (for example in hydrophobicsolvents, which promote an aggregation via polar regions) and to theusage.

This assumption is supported in that B. licheniformis α-amylase, inhaving a comparatively low tendency to multimerization, has asubstantially negative charge potential on its surface.

Another advantageous effect, in addition to avoiding di- and/ormultimerization, is the fact that, from a statistical point of view, theenzymes are mainly in their native conformation and are not deformed bysaid electrostatic interactions which result otherwise in target areasfor said denaturing agents. This effect overall increases theirstability, for example to organic solvents and surfactants which attachto and try to solubilize the hydrophobic areas which are usually lessaccessible to the solvent, or to proteases attacking the internal acidamide bonds of the backbone. Said effect also gives superior protectionto internal oxidizable amino acid residues, for example against oxygenor bleaches.

The object in question is consequently solved by α-amylase variantshaving at least one amino acid substitution over the starting molecule,whereby at least one amino acid residue of the starting molecule, whichis located on the surface of said molecule and makes a neutral orpositively polar or charged contribution to the electrostatic potentialof said molecule, has been replaced with a more negatively polar ornegatively charged amino acid residue, with the following amino acidsubstitutions being possible:

Starting amino acid to give Arg (R) K, Y, C, H, G, A, V, L, I, M, F, W,P, S, T, N, Q, E or D Lys (K) Y, C, H, G, A, V, L, I, M, F, W, P, S, T,N, Q, E or D Tyr (Y) C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or DCys (C) H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D His (H) G, A,V, L, I, M, F, W, P, S, T, N, Q, E or D Gly (G) A, V, L, I, M, F, W, P,S, T, N, Q, E or D Ala (A) V, L, I, M, F, W, P, S, T, N, Q, E or D Val(V) L, I, M, F, W, P, S, T, N, Q, E or D Leu (L) I, M, F, W, P, S, T, N,Q, E or D Ile (I) M, F, W, P, S, T, N, Q, E or D Met (M) F, W, P, S, T,N, Q, E or D Phe (F) W, P, S, T, N, Q, E or D Trp (W) P, S, T, N, Q, Eor D Pro (P) S, T, N, Q, E or D Ser (S) T, N, Q, E or D Thr (T) N, Q, Eor D Asn (N) Q, E or D Gln (Q) E or D Glu (E) D

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Representation of the charge and polarity distribution on theConolly surface of Bacillus sp. A 7-7 (DSM 12368) α-amylase (SEQ ID NO.2). The image was generated with the aid of the Swiss-Pdb viewer (SwissInstitute of Bioinfomatics).

Color Coding

-   -   gray: negative charge or polarization    -   white: neutral charge or polarization    -   black: positive charge or polarization        The surface at the back of the molecule, which is not visible in        this image and which also contains the active site, has a        continuous negative charge pattern.

FIGS. 2A-2C: Alignment of Bacillus sp. A 7-7 (DSM 12368) α-amylase (SEQID NO. 2) with the most important other α-amylases of the prior art, ineach case across the region of the mature protein, i.e. positions 32 to516 according to SEQ ID NO. 2. Individual numbering can be carried outon the basis of the positional number indicated after the particularname of the first amino acid depicted in each case. These other listedα-amylases are S707 (Bacillus sp. #707 (SEQ ID NO. 3)), LAMY (Bacillussp. KSM-Ap1378 (SEQ ID NO. 4)), BAA (Bacillus amyloliquefaciens (SEQ IDNO. 5)), BLA (Bacillus licheniformis (SEQ ID NO. 6)), BstA (Bacillusstearothermophilus (SEQ ID NO. 7)), MK716 (Bacillus sp. MK716 (SEQ IDNO. 8)), TS-23 (Bacillus sp. TS-23 (SEQ ID NO. 9)) and K38 (Bacillus sp.KSM-K38 (SEQ ID NO. 10).

DETAILED DESCRIPTION

α-Amylases mean in accordance with the present application, asillustrated at the outset, the enzymes of class E.C. 3.2.1.1, whichhydrolyze internal α-1,4-glycosidic bonds of starch and starch-likepolymers with the formation of dextrins and β-1,6-branchedoligosaccharides. The invention can in principle be applied to any knownand still-to-be-found α-amylases, as long as they can be homologizedwith the α-amylase of Bacillus sp. A 7-7 (DSM 12368; SEQ ID NO. 2 of thepresent application). Such a homologization is depicted in FIG. 2 forthe industrially most important α-amylases and allows or facilitatesdetecting the amino acid positions according to the invention in theother amylases. The invention is preferably applied to those α-amylaseswhich are already employed successfully in industrial fields. Examplesof α-amylase established in the prior art are given in the introduction.

α-Amylase variants are those α-amylases which have been derived from aprecursor molecule by genetic modifications known per se. “Variant” isthe term at the protein level, which corresponds to “mutant” at thenucleic acid level. The precursor or starting molecules may be wild typeenzymes, i.e. those which are obtainable from natural sources. Thesehave been introduced by way of example at the outset. They may also beenzymes which are already variants per se, i.e. which have already beenmodified compared to the wild type molecules. They mean, for example,point mutants, those with changes in the amino acid sequence, overseveral positions or longer contiguous regions, or else hybrid moleculeswhich are composed of sections of various wild type α-amylases, whichcomplement each other. Wild type enzymes as well as mutants and hybridenzymes have been introduced by way of example at the outset. It ispossible, according to the invention, to further develop in principleany α-amylases. If the nucleic acids encoding them are known, this iscarried out by way of established methods of mutagenesis on the nucleicacids in question; if said nucleic acids are not known, it is possibleto derive, on the basis of the amino acid sequence, nucleic acidsequences coding therefor and to modify the latter accordingly.

Amino acid substitutions mean substitutions of one amino acid by anotheramino acid. According to the invention, such substitutions are indicatedwith reference to the positions in which the substitution takes place,where appropriate in combination with the amino acids in question in theinternationally customary one-letter code. “Substitution in position 83”means, for example, that a variant has a different amino acid in theposition which has position 83 in the sequence of a reference protein.Such substitutions are usually carried out at the DNA level by way ofmutations of individual base pairs (see above). “N83D” means, forexample, that the reference enzyme has the amino acid asparagine atposition 83, while the observed variant has the amino acid aspartic acidat the position homologizable therewith. “83D” means that any, i.e.usually a naturally predetermined, amino acid has been replaced withaspartic acid at a position corresponding to position 83; and “N83X”means that the amino acid asparagine in position 83 has been replacedwith principally any other amino acid.

In principle, the amino acid substitutions according to the inventionand specified by the present application are not limited to the factthat they are the only substitutions in which the variant in questiondiffers from the wild type molecule. It is known in the prior art thatthe advantageous properties of individual point mutations can complementeach other. An α-amylase optimized with respect to particular propertiessuch as, for example, calcium binding or stability to surfactants may bedeveloped according to the invention additionally by the substitutionspresented herein. Therefore, embodiments of the present inventioncomprise any variants which have, in addition to other substitutions,also the substitutions according to the invention, compared to the wildtype molecule.

A reference enzyme with respect to numbering of the positions, which maybe considered according to the invention, is Bacillus sp. A 7-7 (DSM12368) α-amylase whose nucleotide sequence is indicated in SEQ ID NO. 1,followed by the corresponding amino acid sequence in SEQ ID NO. 2. Asillustrated in Example 1 of the present application, this sequenceinformation has been corrected in comparison with the description in WO02/10356 A2 in two positions, and in this form corresponds, according tothe current knowledge, exactly to the sequence data obtainable from thedeposited strain DSM 12368 described in WO 02/10356.

Said correct amino acid sequence is also revealed in the first lines ofthe alignment depicted in FIG. 1, which has been established only forthe mature parts of the particular enzymes. This is justified by thefact that in vivo only the mature portion (i.e. that with the positivenumbering in SEQ ID NO. 2) is active as α-amylase. or some enzymes suchas, for example, AA349 and AA560, the patent literature (WO 00/60060 A2)indicates anyway merely the mature sequence parts.

The surface of the enzyme comprises all those amino acids of thenatively folded enzyme which face the solvent. In Bacillus sp. A 7-7(DSM 12368) α-amylase, for example, these are the 407 amino acids listedin detail in Example 2.

The surface amino acids of other α-amylases may in principle be obtainedby using the alignment of FIG. 2. This is true only approximately,however. Highly conserved and structurally secured regions such asα-helices or β-sheets usually produce good agreement; in more flexibleregions, in particular loops, the comparison based on the alignment,i.e. the primary structure, is uncertain. In all cases (if known), thesecure X-ray structure, as it is actually deposited meanwhile for mostcommercially important α-amylases in generally accessible databases, isdecisive. Said databases are, for example, GenBank (National Center forBiotechnology Information NCBI, National Institutes of Health, Bethesda,Md., USA) and Swiss-Prot (Geneva Bioinformatics (GeneBio) S.A., Geneva,Switzerland).

If the 3D (or tertiary) structure has not yet been determined for amolecule of interest, the former may be obtained via homology modeling.This method of predicting the structure of proteins whose crystalstructures have not yet been resolved assumes that proteins having asimilar primary structure also have similar secondary and tertiarystructures. The similarity of two protein sequences can be determinedvia a suitable algorithm, for example BLAST, FASTA or CLUSTAL. Suchalgorithms are likewise available via generally accessible proteindatabases; thus, for example, GenBank and Swiss-Prot have correspondinglinks. The RSCB protein database (accessible via Max-Delbrück-Zentrum inBerlin, Germany) enables the user to find for a particular sequencecrystal structures of related proteins by way of a FASTA search.

The possible calculations based thereupon, for example with the aid ofthe “Swiss-Pdb Viewer”, are described in the publication “SWISS-Modeland the Swiss-PdbViewer: An environment for comparative proteinmodeling” (1997) by N. Guex and M. C. Peitsch in Electrophoresis, Vol.18, pp. 2714 to 2723. Said viewer and its corresponding manual isaccessible free of charge from the organization Swiss Institute ofBioinformatics (Central Administration, Bâtiment Ecole de Pharmacie—room3041, Université de Lausanne, 1015 Lausanne, Switzerland).Superimposition of said structures can establish a leader structure ontowhich the protein sequence of the α-amylase of interest is then modeled.The individual steps for this can be found in said user manual.

All of the depictions of the surface of an enzyme discussed herein, i.e.the special listing of the surface amino acids of Bacillus sp. A 7-7(DSM 12368) α-amylase (cf. FIG. 1) as well as the modeling approachesillustrated, are based on a calculation. This involves rolling by way ofcalculation a probe of 1.4 Å in size over the surface of said protein.All positions contacted thereby are included in the area of contact withsaid probe and thus in the surface; relatively narrow clefts which aretheoretically open toward the outside are not regarded here as part ofthe surface but are classed as belonging to the interior of themolecule. Using this approach produces the “Connolly surface” which wasfirst described by M. L. Connolly in the article “Solvent-AccessibleSurfaces of Proteins and Nucleic Acids” (1983) in Science, volume 221,709-713. This acknowledged method is familiar to the skilled worker andis used according to the invention for defining the surface ofα-amylases.

In this way, the mentioned 407 surface amino acids were produced forBacillus sp. A 7-7 (DSM 12368) α-amylase, which are listed in detail inExample 2.

The amino acid residues relevant to the invention, which are located onthe surface of the α-amylases contemplated, are those making a neutralor positively polar or charged contribution to the electrostaticpotential of the molecule at pH 7. Such a contribution to theelectrostatic potential of the molecule is defined as the proportion ofthe individual amino acid having charges greater than or equal to zero.

The electrostatic potential at a particular point on the surface isdefined, using the abovementioned Connolly surface, as the potentialwhich acts on the designated probe. This electrostatic potential whichtheoretically is present at any site of the surface may be calculated bysuitable algorithms, and for this purpose the following considerationsare made according to the invention:

The electric field can be considered in principle as the sum of theelectric subfields generated by the individual charges. For a usefulcalculation, the individual charges are considered point charges in afirst approximation. The electric field E_(i) of a point charge Q_(i)can be determined in vacuo at the site r_(i) via the following equation(1), where ∈₀ is the dielectric constant:

$\begin{matrix}{{\overset{\_}{E}}_{i} = {\frac{1}{4\pi \; ɛ_{0}}\frac{Q_{i}}{r_{i}^{2}}{\hat{r}}_{i}}} & (1)\end{matrix}$

A group of N charges thus results in the following electric field E:

$\begin{matrix}{{\overset{\_}{E}}_{i} = {\sum\limits_{i = 1}^{N}\; {\frac{1}{4\pi \; ɛ_{0}}\frac{Q_{i}}{r_{i}^{2}}{\hat{r}}_{i}}}} & (2)\end{matrix}$

The Coulomb potential V_(c)(P₁) generated therefrom at point P₁ in theelectric field E is then defined as follows, where L is the coordinatespace:

V _(c)(P ₁)=∫_(∞) ^(P) ¹ Ē·d L   (3)

This equation can be interpreted in words approximately as follows: theelectrostatic potential at point P₁ is defined as the difference inpotentials of a point at infinity and point P₁. L is a position vectorwhich in practice represents the integration variable of space. PointP₁, and consequently also any other relevant point introduced to thiscalculation in the same manner, is on the protein surface. This formulaenables computers to determine the particular local electrostaticpotential for each point on the protein surface, using suitable computerprograms. As a result, a corresponding charge or polarity can beassigned to each amino acid residue, and this can be illustrated by wayof a visual representation as in FIG. 1, for example.

The calculation using this equation (3) produces an approximation of theelectrostatic potential, which is not accurate but sufficient accordingto the invention. In a first approximation, the dielectric constant ∈₀must be replaced with the actual dielectric constant ∈=∈₀*∈_(r) as thedielectric constant of the medium. This approach is referred to as“Screened Coulomb Potential” and is described by S. A. Hassa et al.(2002) in the article “A Critical Analysis of Continuum Electrostatics:The Screened Coulomb Potential-implicit Solvent Model and the Study ofthe Alanine Dipeptide and Discrimination of Misfolded Structures ofProteins”, in Proteins, volume 45, page 47. Several examples of further,more accurate algorithms can be found in the literature, such as E. L.Mehler et al. (1991), “Electrostatic effects in proteins: comparison ofdielectric and charge models”, Protein Eng., volume 8, pages 903-910,and A. Jakalian et al. (2002), “Fast, efficient generation ofhigh-quality atomic charges. AM1-BCC model: II. Parameterization andvalidation” in J. Comput. Chem., volume 16, pages 1623-1641. These,however, are not required for the application described herein, sinceultimately a semiquantitative statement (positive, neutral, negative)allows the problem to be solved.

The amino acids in question may be calculated, for example, by acomputer algorithm which is available as a further module of the SwissPDB viewer mentioned above. This enables the surface charge distributionof the molecule studied to be calculated by way of the items “tools”,“compute molecular surface” and “electrostatic potential”, with thepartial charges of the atoms of each amino acid, except hydrogen atoms,being taken into account under standard parameters.

As a result of this, 118 residues making a positive or neutralcontribution to the electrostatic potential of the surface weredetermined as a subset of the previously determined 407 surface aminoacid residues of Bacillus sp. A 7-7 (DSM 12368) α-amylase, asillustrated in example 2.

In order to indicate the substitutions allowed in each case, thepolarity values or charges which the various amino acid side chainspossess per se are contemplated. This results in the following order: R,as the most basic and normally positively charged amino acid side chain,followed by K, Y, C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E and D,with D being the most acidic amino acid side chain which carries anegative charge at neutral pH. This explains the grades indicated above,and the structure of the molecule and other charge effects can beassumed according to the invention to play virtually no part in thesubstitution of individual surface amino acids. Thus, if one of saidamino acid side chains were to carry a slightly more negative orslightly more positive partial charge than expected from the well-knownvalues for the free amino acid, then it can be assumed according to theinvention that an amino acid side chain listed further down said ordercarries a correspondingly slightly more negative or slightly morepositive partial charge in the same molecular environment, and the orderstated here of the amino acids allowed for substitution is not alteredoverall.

The illustration in FIG. 1 depicts the pattern, calculated according toexample 2, of different polarities and charges on a surface of Bacillussp. A 7-7 (DSM 12368) α-amylase. The back of the molecule which is notvisible there and which also comprises the active site, has a negativecharge pattern throughout. This explains the relative ease with whichdimers can form in which a molecule having a positively polarized orcharged surface, as depicted in FIG. 1, attaches to the negativelycharged/polarized surface located at the back of another molecule andthereby blocks the latter's active site. It should be possible torecognize such dimers by a molecular weight twice as high and/or anamylase activity half as high in relation to the total proteinconcentration, in each case in comparison with a corresponding solutioncontaining monomers. With increasing attachment of further molecules inthe same orientation, the activity of corresponding solutions shoulddecrease further, until a proportion of the enzymes which has aggregatedenough finally precipitates as an amorphous precipitate and has finallybeen denatured and rendered useless.

From a statistical point of view, α-amylase variants of the inventionshould undergo this process less frequently, according to the number andlocation of the substitutions carried out according to the invention.

In a preferred embodiment, α-amylase variants of the invention are thosein which said amino acid residue has an accessibility of at least 10%,preferably at least 20%, particularly preferably at least 30%, prior toamino acid substitution, wherein said accessibility of the amino acidresidue in question is calculated on a scale from 0% (not accessible tothe solvent) to 100% (present in a hypothetical pentapeptide, GGXGG).

Accessibility of an amino acid residue means according to the invention,how well said residue is accessible to the surrounding solvent (usuallywater), with the molecule being in its natural conformation.Determination of this physicochemical property is based on a scale from0 to 100%, with a value of 0% accessibility meaning that the residue inquestion is not accessible to the solvent, and 100% being theaccessibility possible in a hypothetical pentapeptide GGXGG.

According to the invention, this molecular property is reflected in thatan exposed amino acid residue which stands out from the surface contactsother molecules more readily than one which is less accessible to thesolvent. Such residues are therefore preferred targets for theconversion according to the present invention.

Using the abovementioned SwissPDB Viewer enables these values also to becalculated for each surface amino acid of a globular protein.Accessibility of the amino acids is calculated there via the menu item“Select->Accessible aa”, followed by entering the accessibility inpercent. Subsequently, the corresponding amino acids are selected andcan be displayed by pressing the “Return” key. In addition, it ispossible here to automate said calculation via a self-build program, andthis is particularly recommended, if more amino acids are to be includedin said calculation.

As illustrated in Example 2, the following 97 amino acid residues ofBacillus sp. A 7-7 (DSM 12368) α-amylase were determined, which make acontribution to the electrostatic potential of the surface and at thesame time have a value for accessibility of at least 10%, the values forsolvent accessibility being indicated in % in brackets following thepositions listed: T5 (39), N6 (12), G7 (13), N19 (28), N22 (28), N25(16), R26 (27), R28 (39), S29 (38), S32 (28), N33 (28), K35 (37), K37(18), Q53 (12), K72 (27), V75 (30), R76 (24), T81 (16), R82 (22), N83(44), Q84 (24), Q86 (18), A87 (18), T90 (30), A91 (11), K93 (32), S94(50), N95 (19), G96 (25), Q98 (29), R118 (41), T136 (22), K142 (30),G149 (14), N150 (39), T151 (22), H152 (27), N154 (41), K156 (30), R158(33), Y160 (20), R171 (32), Q172 (53), R176 (41), R181 (34), R218 (18),T227 (19), G229 (14), K242 (15), R247 (20), T251 (23), R254 (15), K259(26), N260 (49), K281 (33), N283 (40), R302 (50), R310 (31), R320 (52),T323 (49), R359 (13), Y368 (12), Y372 (37), T376 (56), K383 (19), K385(37), Q394 (20), K395 (38), G399 (16), K400 (44), Y404 (11), G417 (11),N418 (25), T419 (59), A420 (37), H421 (16), P422 (46), G435 (25), G436(17), W439 (47), R444 (49), N445 (41), Q449 (31), V450 (24), R452 (33),R458 (24), S459 (52), G460 (32), T461 (35), T463 (40), N465 (22), A466(37), N471 (20), S473 (10), N475 (25), G476 (31), N484 (12).

The further preferred embodiments of this molecule with respect toaccessibility can be selected on the basis of the specified percentageaccessibility.

An application to other α-amylases is possible by way of approximationvia an alignment such as in FIG. 2. However, as illustrated previously,the actual three dimensional structure is decisive, on the basis ofwhich the appropriate calculations of actual charge distribution andsolvent accessibility can be carried out, as demonstrated for Bacillussp. A 7-7 (DSM 12368) α-amylase in the example.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants, wherein said amino acid residue is locatedin a neutral or positively polarized or charged region consisting of atleast two directly adjacent amino acid residues on the surface.

This thus involves carrying out the calculation illustrated above andselecting for the mutagenesis of the invention an amino acid of the kindthat, in addition to the abovementioned properties, is immediatelyadjacent to just such a residue on the surface, i.e. it is notsurrounded exclusively by those amino acid residues which cannot alsoundergo a substitution according to the invention.

Thus, an amino acid of the kind that is not on its own but part of aneutral or positively polar or charged surface of the molecule is herebysubjected to a mutation according to the invention. This breaks thetwo-dimensional arrangement which, as explained above, may in particularbe regarded as the cause for α-amylases to aggregate via electrostaticinteractions and form multimers. From a statistical point of view, anisolated positive charge or polarity should make a smaller contributionto multimerization, since it should have a smaller effect on othermolecules due to increasingly more negatively charged adjacent aminoacids. Therefore preference is given to carrying out substitutions incontiguous regions which appear neutral or positive. These are depictedin black or white for the example of Bacillus sp. A 7-7 (DSM 12368)α-amylase in FIG. 1.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants, wherein the amino acid residue to bemutated according to the invention is located in a position belonging toeither of the following two groups:

-   (A) 5, 6, 7, 19, 22, 25, 26, 28, 29, 32, 33, 35, 37, 53, 72, 75, 76,    81, 82, 83, 84, 86, 87, 90, 91, 93, 94, 95, 96, 98, 118, 136, 142,    149, 150, 151, 152, 154, 156, 158, 160, 171, 172, 181, 227, 229,    247, 251, 254, 259, 260, 281, 283, 394, 395, 399, 400, 417, 418,    419, 420, 421 and 422; or-   (B) 435, 436, 439, 444, 445, 449, 450, 452, 458, 459, 460, 461, 463,    465, 466, 471, 473, 475, 476 and 484,    in each case indicated in the numbering of the mature protein    according to SEQ ID NO. 2.

As described in example 3, it was possible to accurately model thesurface charge distribution of Bacillus sp. A 7-7 (DSM 12368) α-amylase.It turned out that it is possible to classify the 97 amino acid residueslocated on the surface of the molecule and making a neutral orpositively polar or charged contribution to the electrostatic potentialof said molecule and additionally having an accessibility of more than10% into three groups depending on their location. In this connection,the residues of groups A and B are in each case contiguous regions ofneutral or positive polarity or charge.

Group A refers to the following 63 amino acid positions, wherein in eachcase the amino acid present there in Bacillus sp. A 7-7 (DSM 12368)α-amylase is indicated:

T5, N6, G7, N19, N22, N25, R26, R28, S29, S32, N33, K35, K37, Q53, K72,V75, R76, T81, R82, N83, Q84, Q86, A87, T90, A91, K93, S94, N95, G96,Q98, R118, T136, K142, G149, N150, T151, H152, N154, K156, R158, Y160,R171, Q172, R181, T227, G229, R247, T251, R254, K259, N260, K281, N283,Q394, K395, G399, K400, G417, N418, T419, A420, H421, P422.

Group B includes the following 20 positions:

G435, G436, W439, R444, N445, Q449, V450, R452, R458, S459, G460, T461,T463, N465, A466, N471, S473, N475, G476, N484.

The remaining 14 amino acid residues which form group C may beconsidered neutral or positive islands within otherwise negativepolarized or charged regions:

R176 (41), R218 (18), K242 (15), R302 (50), R310 (31), R320 (52), T323(49), R359 (13), Y368 (12), Y372 (37), T376 (56), K383 (19), K385 (37),Y404 (11).

The last-mentioned amino acid residues can be assumed to make a rathersmall contribution to the aggregation tendency of the whole molecule.The contiguous regions A and B should cause multimerization all the morebecause they should exert a stronger electrostatic effect which causesthe arrangement of a plurality of molecules and which, as assumedaccording to the invention, results in the observed tendency toaggregate. Therefore preference is given to carrying out the amino acidsubstitutions of the invention in said contiguous regions A and/or B.

The same considerations and calculations as in the example of Bacillussp. A 7-7 (DSM 12368) α-amylase may be carried out in principle for anyother α-amylase established in the prior art. Mention should be made, inparticular in the case of these specific positions, of the more simplecomparison of the corresponding amino acid sequences via an alignment.In order to implement the aspect of the invention described herein, theparticular α-amylases can be homologized with Bacillus sp. A 7-7 (DSM12368) α-amylase. Such an alignment is indicated, for example, in FIG. 2for the most important α-amylases established in the prior art; thenumbering of the mature protein according to SEQ ID NO. 2 corresponds toline 1 in FIG. 2. The positions listed here, which are preferred for thesubstitutions of the invention, can be derived by way of such analignment for the enzyme of interest in each case and can be modifiedaccording to the invention with at least approximately the same successas for the enzyme looked at herein. As explained above, however, thespecific situation in the enzyme in question is ultimately decisive ineach case.

In a further preferred embodiment, the α-amylase variants of theinvention are those α-amylase variants, wherein a plurality of saidamino acid substitutions have been carried out, preferably at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30, particularly preferably between 10 and30, and very particularly preferably between 15 and 25.

This is because the solution to the present object is based on theobservation that some α-amylases form amorphous aggregates, and theexistence of relatively large, differently charged or polarizedtwo-dimensional regions was considered a possible explanation for this.At the same time, α-amylases having mainly negative surface charges areknown in the prior art. It is therefore advantageous to convertaccording to the invention even more than only one residue to a morenegatively polar or negatively charged amino acid residue.

Due to the nature of the enzymes, this procedure also has an individualupper limit which must be determined experimentally, where appropriate.Thus, a molecule that is covered with too many negative charges islikely to be repelled too much by the substrate and thereby loseactivity. Moreover, too many charges may also be disadvantageous for thefolding so that, from a critical value upward, too many misfolded, andtherefore non-active, molecules would be formed.

At least for Bacillus sp. A 7-7 (DSM12368) α-amylase, it has thereforeproved to be advantageous to limit the number of substitutions accordingto the invention to less than 30, of which more than 10 can be regardedas really advantageous. A similar result can be expected for theα-amylases homologizable therewith, for example those of FIG. 2 and inparticular those which likewise bear neutral or positively charged orpolarized amino acids in the homologous positions.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants in which in from at least one to no morethan exactly as many other positions one or more other amino acidresidues on the surface of the starting molecule have been replaced withless negatively polar or negatively charged amino acid residues, with ineach case the following amino acid substitutions being possible:

Starting amino acid to give K, Y, C, H, G, A, V, L, I, M, F, W, P, S, T,N, Q, E or D Arg (R) Y, C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, Eor D Lys (K) C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D Tyr (Y)H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D Cys (C) G, A, V, L, I,M, F, W, P, S, T, N, Q, E or D His (H) A, V, L, I, M, F, W, P, S, T, N,Q, E or D Gly (G) V, L, I, M, F, W, P, S, T, N, Q, E or D Ala (A) L, I,M, F, W, P, S, T, N, Q, E or D Val (V) I, M, F, W, P, S, T, N, Q, E or DLeu (L) M, F, W, P, S, T, N, Q, E or D Ile (I) F, W, P, S, T, N, Q, E orD Met (M) W, P, S, T, N, Q, E or D Phe (F) P, S, T, N, Q, E or D Trp (W)S, T, N, Q, E or D Pro (P) T, N, Q, E or D Ser (S) N, Q, E or D Thr (T)Q, E or D Asn (N) E or D Gln (Q) D Glu (E)

This mutation which runs counter to the actual invention attenuatesagain the charge effect introduced according to the invention. Thispursues two aims: firstly, as mentioned above, it is possible, inparticular if a plurality of mutations of the invention are carried out(up to 30 are considered particularly sensible according to theinvention), for the α-amylase molecule to receive a negative chargewhich is too high overall and which therefore could impair stability andreactivity. In this respect, in particular if a plurality of mutationsare carried out, it may be reasonable for the overall charge effect tobe attenuated again.

Secondly, this breaks the two-dimensionality of the charge distributionon the surface of the enzyme. As illustrated above, saidtwo-dimensionality was considered an essential reason formultimerization. If the charge pattern is altered to give a looserpattern with overall the same or only slightly changed total charge, thetendency to aggregate can likewise be assumed to decrease because themolecules do no longer align themselves automatically in a regularorientation, like magnets.

The substitutions allowed in each case are designated by falling back tothe reflection illustrated above on the polarity values or charges whichthe various amino acid side chains possess per se. This results in theexactly reverse order because R, as the most basic and normallypositively charged amino acid side chain, cannot be replaced with anyother to cause a more positive polarity or charge, but can itselfreplace any other side chain. This applies, in a corresponding grading,to K, Y, C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E and D, with Dbeing the most acidic amino acid side chain which normally carries anegative charge at neutral pH and which can be replaced with any otherside chain, including E, under this aspect of the invention.

In a further preferred embodiment, the α-amylase variants of theinvention are those α-amylase variants, wherein the substitution(s)carried out in order to reduce the change in the overall charge havebeen carried out at amino acid position(s) which does/do not belong tothe following:

-   (A) 5, 6, 7, 19, 22, 25, 26, 28, 29, 32, 33, 35, 37, 53, 72, 75, 76,    81, 82, 83, 84, 86, 87, 90, 91, 93, 94, 95, 96, 98, 118, 136, 142,    149, 150, 151, 152, 154, 156, 158, 160, 171, 172, 181, 227, 229,    247, 251, 254, 259, 260, 281, 283, 394, 395, 399, 400, 417, 418,    419, 420, 421 and 422 or-   (B) 435, 436, 439, 444, 445, 449, 450, 452, 458, 459, 460, 461, 463,    465, 466, 471, 473, 475, 476 and 484 or-   (C) 176, 218, 242, 302, 310, 320, 323, 359, 368, 372, 376, 383, 385    and 404, in each case indicated in the numbering of the mature    protein according to SEQ ID NO. 2.

This preferred embodiment makes use of the finding for Bacillus sp. A7-7 (DSM 12368) α-amylase (SEQ ID NO. 2), according to which these aminoacid residues which can be assigned to regions A and B form large areasof neutral or positively polarized or charged regions, and the residueslisted under (C) form neutral or positively polarized or charged islandswithin a mainly negative environment. Since, as illustrated above, theaspect of the invention contemplated here intends to break especiallythe two-dimensionality of charge distribution, it is particularly usefulto select positions other than the ones designated herein for thecharge-leveling back mutation. This is because this would again enlargethe neutrally or positively polarized or charged regions specified.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants wherein the starting molecule is any of thefollowing α-amylases: α-amylase from Bacillus sp. A 7-7 (DSM 12368) (SEQID NO. 2), α-amylase from Bacillus sp. #707 (SEQ ID NO. 3), α-amylasefrom Bacillus sp. KSM-AP1378 (SEQ ID NO. 4), α-amylase from Bacillus sp.KSM-K38 (SEQ ID NO. 10), α-amylase from B. amyloliquefaciens (SEQ ID NO.5), α-amylase from B. licheniformis (SEQ ID NO. 6), α-amylase fromBacillus sp. MK716 (SEQ ID NO. 8), α-amylase from Bacillus sp. TS-23(SEQ ID NO. 9), α-amylase from B. stearothermophilus (SEQ ID NO. 7),α-amylase from B. agaradherens, a cyclodextrin glucanotransferase(CGTase) from B. agaradherens, in particular from B. agaradherens (DSM9948), or a hybrid amylase therefrom and/or an α-amylase derivedtherefrom by mutagenesis of single or multiple amino acids.

As illustrated at the outset, numerous α-amylases are established in theprior art for the use for various industrial purposes. These include,for example, fungal and bacterial enzymes. The present invention may inprinciple be applied to all of these α-amylases.

Those α-amylases of gram-positive bacteria, in particular of the genusBacillus, which are adapted to an alkaline environment, are particularlycommon for industrial purposes. This is because they have favorableproperties for these fields of use from the outset. Accordingly, thepresent invention is directed in particular to the α-amylases which canbe obtained naturally from said species. These include in particular thefollowing:

-   -   Bacillus sp. A 7-7 (DSM 12368) α-amylase, disclosed in WO        02/10356 A2 and the present application (SEQ ID NO. 2);    -   Bacillus sp. #707 α-amylase (SEQ ID NO. 3), disclosed in the        publication

“Nucleotide sequence of the maltohexaose-producing amylase gene from analkalophilic Bacillus sp. #707 and structural similarity to liquefyingtype α-amylases” (1988) by Tsukamoto et al., Biochem. Biophys. Res.,Comm., Vol. 151(1), pp. 25-31;

-   -   Bacillus sp. KSM-AP1378 α-amylase (SEQ ID NO. 4), whose amino        acid sequence (together with point mutants) has been disclosed        in EP 985731 A1;    -   Bacillus sp. KSM-K38 α-amylase (SEQ ID NO. 10), disclosed in EP        1022334 A2;    -   B. amyloliquefaciens α-amylase (SEQ ID NO. 5) (commercial        product BAN® from Novozymes);    -   B. licheniformis α-amylase (SEQ ID NO. 6) (commercial product        Termamyl® from Novozymes);    -   Bacillus sp. MK716 α-amylase (SEQ ID NO. 8), whose sequence has        been deposited under number AAB18785 in the database of the        National Center for Biotechnology Information, National Library        of Medicine, Building 38A, Bethesda, Md. 20894, USA (GenBank);    -   Bacillus sp. TS-23 α-amylase (SEQ ID NO. 9), disclosed in the        publication: “Production and property of raw-starch-degrading        Amylase from the thermophilic and alkaliphilic Bacillus sp.        TS-23” (1998) by Lin et al. in Biotechnol. Appl. Biochem., Vol.        28(1), pp. 61 to 68; and    -   B. stearothermophilus α-amylase (SEQ ID NO. 7) (commercial        product Novamyl® from Novozymes).        The amino acid sequences comprising the in each case mature        regions of these enzymes are compiled in FIG. 2.

The present invention further relates to B. agaradherens α-amylase andtwo B. agaradherens cyclodextrin glucanotransferases (CGTases), whichare described in two international applications: WO 02/06508 A2 and WO02/44350 A2, the latter being formed by the deposited microorganism B.agaradherens (DSM 9948) described in the application in question andbeing preferred. The part of the CGTase molecule, which can behomologized with the previously described α-amylases, is in each casemodified according to the invention.

The starting enzyme to be introduced to the invention may also be ahybrid amylase of the α-amylases just mentioned. These are revealed, asalready mentioned at the outset, for example by the applications WO96/23784 A1 (hybrids of the α-amylases of Bacillus licheniformis (SEQ IDNO. 6), B. amyloliquefaciens (SEQ ID NO. 5) and B. stearothermophilus(SEQ ID NO. 7)) and WO 03/014358 A2 (special hybrid amylases of Bacilluslicheniformis (SEQ ID NO 6) and B. amyloliquefaciens (SEQ ID NO. 5)).The latter will be discussed in more detail hereinbelow.

In addition, the prior art describes numerous α-amylase variants derivedby mutagenesis of single or multiple amino acids of said α-amylases. Thepresent invention may likewise be applied to all of these, and therespective effects can be assumed in principle to be additive. Thus itshould be possible, by carrying out a mutation of the invention, toimprove an α-amylase variant which is particularly powerful in a specialfield of application, owing to a particular amino acid substitution,also in its tendency to aggregate, in addition to the former aspect ofits performance. These are thus preferably amino acid variations forwhich a corresponding advantage has been described previously.

In this connection, it does not matter in principle, in which order thesubstitutions in question have been carried out, i.e. whether acorresponding point mutant is further developed according to theinvention or initially a variant of the invention is generated, forexample, from a wild type molecule and is then further developedaccording to other teachings which can be found in the prior art. It isalso possible to carry out a plurality of substitutions, for examplethose according to the invention and others together, in a singlemutagenesis mix simultaneously. This situation is given, for example, ifan α-amylase is further developed using randomly acting mutagenesisprocesses, for example by means of mutagenizing agents or shuffling.This applies to any type of modification of the enzymes in question, inparticular to point mutations which in principle act independently ofone another.

Particularly powerful α-amylases obtainable by point mutations arerevealed, for example, by the application WO 00/60060 A2 whichdescribes, on the basis of the AA560 amylase (the same numbering as themature protein in SEQ ID NO. 2), numerous mutations in positions 181,182, 183, 184, 195, 206, 212, 216, and 269. This application can beconsidered a development of WO 96/23873 A1 which had previouslydescribed the possibility of point mutagenesis in positions 180, 181,182, 183, 184 and 185 in order to improve performance. The applicationWO 00/60059 A2 specifies further improvements of enzymes referred totherein as Termamyl-like amylases; according to this, positions 13, 48,49, 50, 51, 52, 53, 54, 57, 107, 108, 111, 168 and 197 (according to thenumbering of B. licheniformis α-amylase) are said to be suitablestarting points for this. All of these α-amylases which are improvedwith respect to their activity and/or performance via these positionsand in particular according to said applications, are, for the purposesof the present application, starting molecules particularly preferredaccording to the invention.

In particularly preferred embodiments, the starting molecule is any ofthe following α-amylases: α-amylase from Bacillus sp. A 7-7 (DSM 12368)(SEQ ID NO. 2), cyclodextrin glucanotransferase from B. agaradherens(DSM 9948) or a hybrid amylase of the α-amylases from B.amyloliquefaciens (SEQ ID NO. 5) and from B. licheniformis (SEQ ID NO.6), preferably a hybrid amylase AL34, AL76, AL112, AL256, ALA34-84,LAL19-153 or LAL19-433.

Thus the invention described herein is illustrated in the examples ofthe present application in particular on the basis of Bacillus sp. A 7-7(DSM 12368) α-amylase (SEQ ID NO. 2), whose most likely correct DNA andamino acid sequences are indicated in the sequence listing of thepresent application. This wild type enzyme has the advantagesillustrated in the application WO 02/10356 A2 with regard to a use indetergents and cleansers over other established α-amylases. Anotherparticularly preferred starting molecule, namely B. agaradherens (DSM9948) cyclodextrin glucanotransferase, has already been mentioned aboveand likewise makes particular contributions to the overall performanceof detergents and cleansers over other enzymes, as described in theapplication WO 02/44350 A2.

The application WO 03/014358 describes hybrid amylases of the α-amylasesB. amyloliquefaciens and B. licheniformis. Of these, the moleculesreferred to by the acronyms AL34, AL76, AL112, AL256, ALA34-84,LAL19-153 or LAL19-433 have exhibited particularly advantageousperformances when used in detergents and cleansers and are thereforeparticularly preferably also introduced to the present invention, i.e.are mutated according to the invention. The particular names refer tothe elements of which they are composed in each case, as seen from the Nterminus, wherein A is the B. amyloliquefaciens α-amylase and L is theB. licheniformis α-amylase and the subsequent number indicates thejunction between the first and second amino acid sequences. Furtherdetails on this can be found in said application.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants, wherein the starting molecule is anα-amylase whose amino acid sequence is at least 96% identical,preferably 98%, particularly preferably 100%, identical to the aminoacid sequence indicated in SEQ ID NO. 2 in positions +1 to 484.

This Bacillus sp. A 7-7 (DSM 12368) α-amylase is described in detail inthe international application WO 02/10356 A2 which has already beenmentioned several times. It should be noted in this context that thenucleotide sequence and the amino acid sequence derived therefrom of theα-amylase obtainable from said strain have most likely the sequencesindicated in SEQ ID NO. 1 and 2, respectively, of the presentapplication. This represents a correction of the sequence indicated inthe application WO 02/10356 A2. The experiments described in thatapplication have also been carried out using the native α-amylaseobtainable from said strain.

The nucleotide sequence according to SEQ ID NO. 1 enables the skilledworker immediately to carry out mutations according to the invention. Anα-amylase-encoding DNA which has been prepared according to the sequencelisting of WO 02/10356 A2 rather than by using the depositedmicroorganism, may be converted with the aid of simple point mutagenesismethods, as they are also mentioned in the examples of the presentapplication, to a nucleotide sequence according to SEQ ID NO. 1 of thepresent application.

The possibilities of culturing the corresponding microorganism,purification and enzymic characterization of the wild type α-amylasehave likewise been disclosed in that application and are summarizedagain in Example 1 of the present application. Variants according to theinvention of this enzyme can in principle be produced and purified inthe same way, with the differences introduced according to theinvention, for example in view of the isoelectric point (IEP), having tobe taken into account. For example, the IEP could be shifted into theacidic range somewhat.

Further preference is given to those α-amylase variants according to theinvention, wherein the starting molecule is an α-amylase variantcontaining, with increasing preference, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or11 additional point mutations.

The sequences indicated in SEQ ID NO. 1 and 2 differ in each case in twopositions from the information in WO 02/10356 A2, as illustrated inExample 1. According to current knowledge, they represent the bestdescriptions of Bacillus sp. A 7-7 (DSM 12368) α-amylase and aretherefore particularly preferred starting points for introducing aminoacid substitutions of the invention.

According to the above, it may further be assumed that in principle anyα-amylase can be improved by individual point mutations, i.e. deletions,insertions or substitutions of individual amino acids, with respect toother aspects. Thus, as already illustrated at the outset, theapplications WO 00/22103 A1, WO 96/23873 A1, WO 00/60060 A2 and WO01/66712 A2 reveal α-amylase variants which have been mutagenized inindividual positions and which have been improved in each case withrespect to special aspects. Since some of said variants have highhomology to said amylase depicted in SEQ ID NO. 2, i.e. they can bereferred to as highly related, the substitutions specified therein canbe expected to be applicable also to these molecules and, analogously,also to other α-amylases, with the same advantageous effects. Furtherpossible modifications can be found in the prior art as summarized, forexample, at the outset of the present application.

Furthermore preferred embodiments of the present invention are α-amylasevariants according to the invention, having the additional pointmutations in one or more of the following positions: 5, 167, 170, 177,202, 204, 271, 330, 377, 385 and 445, according to the numbering of themature protein according to SEQ ID NO. 2.

Thus, the German application DE 10309803.8 which has not beenprepublished reveals that point mutations may be carried out inpositions −19, 5, 167, 170, 177, 204, 271, 330, 377, 385 and/or 445 (or13, 32, 194, 197, 203, 230, 297, 356, 406, 414 and 474 according to thenumbering of the unprocessed B. amyloliquefaciens α-amylase), in orderto improve the alkali activity of α-amylases. Preference is thereforegiven to applying the present invention to these already improvedmolecules.

The application WO 94/18314 A1 reveals α-amylase variants stabilized tooxidation, including especially in position 197 (according to thenumbering of B. licheniformis α-amylase) corresponding to position 202of the α-amylase according to SEQ ID NO. 2. Variants according to theinvention may be stabilized additionally to oxidation by thesubstitutions mentioned therein, especially in position 202. Saidsubstitution is of particular interest also because M202 in Bacillus sp.A 7-7 (DSM 12368) α-amylase is the only methionine residue which is morethan 10% accessible to the solvent, having an accessibility value of 30%(cf. Example 2). This explains its particular sensitivity to oxidationand the connection with the present invention.

Preferred embodiments thereof are those α-amylase variants, wherein thepoint mutations in the starting enzymes are as follows: 5A, 167R, 170P,177L, 202L, 204V, 271 D, 330D, 377R, 385S and/or 445Q.

Thus, the mentioned application DE 10309803.8 describes the sequencevariations −19P, 5A, 167R, 170P, 177L, 204V, 271D, 330D, 377R, 385Sand/or 445Q (or, according to the numbering of the unprocessed B.licheniformis α-amylase, the substitutions to give 13P, 32A, 194R, 197P,203L, 230V, 297D, 356D, 406R, 414S and 474Q) as preferred substitutions.

WO 94/18314 A1 illustrates in particular the oxidation-stabilizingaction of the substitution M197L corresponding to the amino acidsubstitution M202L according to SEQ ID NO. 2. Variants according to theinvention may therefore be stabilized additionally to oxidationparticularly by this point mutation.

Another subject matter of the present invention are methods ofincreasing the stability of an α-amylase to a dimerization and/ormultimerization brought about by electrostatic interactions, whereby atleast one amino acid residue on the surface of the starting molecule,which makes a neutral or positively polar or charged contribution to theelectrostatic potential of said molecule, is replaced with a morenegatively polar or negatively charged amino acid residue, with thefollowing amino acid substitutions being possible:

Starting amino acid to give Arg (R) K, Y, C, H, G, A, V, L, I, M, F, W,P, S, T, N, Q, E or D Lys (K) Y, C, H, G, A, V, L, I, M, F, W, P, S, T,N, Q, E or D Tyr (Y) C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or DCys (C) H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D His (H) G, A,V, L, I, M, F, W, P, S, T, N, Q, E or D Gly (G) A, V, L, I, M, F, W, P,S, T, N, Q, E or D Ala (A) V, L, I, M, F, W, P, S, T, N, Q, E or D Val(V) L, I, M, F, W, P, S, T, N, Q, E or D Leu (L) I, M, F, W, P, S, T, N,Q, E or D Ile (I) M, F, W, P, S, T, N, Q, E or D Met (M) F, W, P, S, T,N, Q, E or D Phe (F) W, P, S, T, N, Q, E or D Trp (W) P, S, T, N, Q, Eor D Pro (P) S, T, N, Q, E or D Ser (S) T, N, Q, E or D Thr (T) N, Q, Eor D Asn (N) Q, E or D Gln (Q) E or D Glu (E) D

According to the invention, the tendency to form amorphous α-amylaseaggregates is understood as meaning the formation of dimers, trimers oraggregates from more individual α-amylase molecules. This is understoodas being a stability aspect, since this causes the enzyme preparation inquestion from a macroscopic point of view, as illustrated at the outset,to lose a considerable proportion of activity. Any method thatcounteracts aggregation is therefore one that increases the stability ofthe enzyme preparation in question with respect to its overall activity.

The approach according to the invention in order to solve said problemconsists of, as likewise illustrated above, restricting themultimerization brought about by electrostatic interactions. This iscarried out by altering the charges and polarity carriers on theα-amylase surface in the direction of less neutral or less positivepolarities, i.e. to rather negative polarities and charges. Preferenceis given here, as illustrated above and reiterated hereinbelow, tosubstitutions in particular regions and particular positions.

Methods of preparing enzyme variants are known per se to abiotechnologist. He will use in particular the nucleic acids coding forthe starting enzymes in question, which is mutated in the correspondingcodon according to the amino acid to be introduced.

The principle of this is also illustrated in Example 3: primers whichcover the region to be mutated and contain the mutation to be introduced(mismatch primers) are synthesized, for example, with the aid of theQuikChange kit (Stratagene, cat. NO. 200518) according to thecorresponding protocol. The primer sequences are designed on the basisof the corresponding nucleotide sequence, taking into account theuniversal genetic code. In order to generate a less neutral or positivepolarity or charge as already discussed above, the correspondinglygraded amino acid substitutions are possible in this context. Accordingto this principle, an expression vector containing the α-amylasesequence is suitably mutagenized accordingly and transformed into anexpression strain suitable for expressing said amylase, using generallyknown methods. Since the variant usually has only a few substitutionscompared to the starting molecule, the systems established for saidstarting molecule can be used for information when choosing theexpression system, culturing and work-up methods. The fact that theelectrostatic properties of α-amylases are altered according to theinvention must be taken into account here. A corresponding change in theIEP may be checked, for example, with the aid of isoelectric focusing.

Other than that, the comments made above regarding the particularenzymes apply accordingly. This also applies accordingly to thepreferred embodiments, i.e. to those methods of the invention which areused to obtain the above-described variants according to the invention.

According to the above, preferred methods of the invention are those,wherein said amino acid residue has an accessibility of at least 10%,preferably at least 20%, particularly preferably at least 30%, prior toamino acid substitution, wherein said accessibility of the amino acidresidue in question is calculated on a scale from 0% (not accessible tothe solvent) to 100% (present in a hypothetical pentapeptide, GGXGG).

According to the above, further preferred methods of the invention arethose, wherein said amino acid residue is located in a neutral orpositively polarized or charged region consisting of at least twodirectly adjacent amino acids residues on the surface.

In this context and according to the above, preference is given tomethods, wherein said amino acid residue is in a position belonging toeither of the following two groups:

(A) 5, 6, 7, 19, 22, 25, 26, 28, 29, 32, 33, 35, 37, 53, 72, 75, 76, 81,82, 83, 84, 86, 87, 90, 91, 93, 94, 95, 96, 98, 118, 136, 142, 149, 150,151, 152, 154, 156, 158, 160, 171, 172, 181, 227, 229, 247, 251, 254,259, 260, 281, 283, 394, 395, 399, 400, 417, 418, 419, 420, 421 and 422;or

(B) 435, 436, 439, 444, 445, 449, 450, 452, 458, 459, 460, 461, 463,465, 466, 471, 473, 475, 476 and 484,

in each case indicated in the numbering of the mature protein accordingto SEQ ID NO. 2.

According to the above, further preferred methods of the invention arethose, wherein a plurality of said amino acid substitutions are carriedout, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30,particularly preferably between 10 and 30 and very particularlypreferably between 15 and 25.

According to the above, further preferred methods of the invention arethose, according to which in from at least one to no more than exactlyas many other positions one or more other amino acid residues on thesurface of the starting molecule have been replaced with less negativelypolar or negatively charged amino acid residues, with in each case thefollowing amino acid substitutions being possible:

Starting amino acid to give K, Y, C, H, G, A, V, L, I, M, F, W, P, S, T,N, Q, E or D Arg (R) Y, C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, Eor D Lys (K) C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D Tyr (Y)H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D Cys (C) G, A, V, L, I,M, F, W, P, S, T, N, Q, E or D His (H) A, V, L, I, M, F, W, P, S, T, N,Q, E or D Gly (G) V, L, I, M, F, W, P, S, T, N, Q, E or D Ala (A) L, I,M, F, W, P, S, T, N, Q, E or D Val (V) I, M, F, W, P, S, T, N, Q, E or DLeu (L) M, F, W, P, S, T, N, Q, E or D Ile (I) F, W, P, S, T, N, Q, E orD Met (M) W, P, S, T, N, Q, E or D Phe (F) P, S, T, N, Q, E or D Trp (W)S, T, N, Q, E or D Pro (P) T, N, Q, E or D Ser (S) N, Q, E or D Thr (T)Q, E or D Asn (N) E or D Gln (Q) D Glu (E)

It is in principle unimportant here, whether the substitution accordingto the invention or this charge-leveling substitution is carried outfirst.

In this context and according to the above, preference is given tomethods, wherein the substitution(s) carried out in order to reduce thechange in the overall charge have been carried out at amino acidposition(s) which does/do not belong to the following:

-   (A) 5, 6, 7, 19, 22, 25, 26, 28, 29, 32, 33, 35, 37, 53, 72, 75, 76,    81, 82, 83, 84, 86, 87, 90, 91, 93, 94, 95, 96, 98, 118, 136, 142,    149, 150, 151, 152, 154, 156, 158, 160, 171, 172, 181, 227, 229,    247, 251, 254, 259, 260, 281, 283, 394, 395, 399, 400, 417, 418,    419, 420, 421 and 422 or-   (B) 435, 436, 439, 444, 445, 449, 450, 452, 458, 459, 460, 461, 463,    465, 466, 471, 473, 475, 476 and 484 or-   (C) 176, 218, 242, 302, 310, 320, 323, 359, 368, 372, 376, 383, 385    and 404, in each case indicated in the numbering of the mature    protein according to SEQ ID NO. 2.

According to the above, further preferred methods of the invention arethose wherein the starting molecule is any of the following α-amylases:α-amylase from Bacillus sp. A 7-7 (DSM 12368) (SEQ ID NO. 2), α-amylasefrom Bacillus sp. #707 (SEQ ID NO. 3), α-amylase from Bacillus sp.KSM-AP1378 (SEQ ID NO. 4), α-amylase from Bacillus sp. KSM-K38 (SEQ IDNO. 10), α-amylase from B. amyloliquefaciens (SEQ ID NO. 5), α-amylasefrom B. licheniformis (SEQ ID NO. 6), α-amylase from Bacillus sp. MK716(SEQ ID NO. 8), α-amylase from Bacillus sp. TS-23 (SEQ ID NO. 9),α-amylase from B. stearothermophilus (SEQ ID NO. 7), α-amylase from B.agaradherens, a cyclodextrin glucanotransferase (CGTase) from B.agaradherens, in particular, from B. agaradherens (DSM 9948), or ahybrid amylase therefrom and/or an α-amylase derived therefrom bymutagenesis of single or multiple amino acids.

Among these, preference is given according to the above two methods,wherein the starting molecule is any of the following α-amylases:α-amylase from Bacillus sp. A 7-7 (DSM 12368) (SEQ ID NO. 2),cyclodextrin glucanotransferase from B. agaradherens (DSM 9948) or ahybrid amylase of the α-amylases from B. amyloliquefaciens (SEQ ID NO.5) and from B. licheniformis (SEQ ID NO. 6), preferably a hybrid amylaseAL34, AL76, AL112, AL256, ALA34-84, LAL19-153 or LAL19-433.

According to the above, further preferred methods of the invention arethose, wherein the starting molecule is an α-amylase whose amino acidsequence is at least 96% identical, preferably 98%, particularlypreferably 100%, identical to the amino acid sequence indicated in SEQID NO. 2 in positions +1 to 485.

Among these, preference is given according to the above to methods,wherein the starting molecule is an α-amylase variant containing, withincreasing preference, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 additionalpoint mutations.

According to the above, further preferred methods of the invention arethose, wherein the introduced α-amylase is an α-amylase variant havingthe additional point mutations in one or more of the followingpositions: 5, 167, 170, 177, 202, 204, 271, 330, 377, 385 and 445,according to the numbering of the mature protein according to SEQ ID NO.2.

At this point, it should be noted once again that it does not matter inprinciple for mutants which can be used as starting molecules accordingto the invention, as to whether the amino acid substitution according tothe invention takes place after or before introducing the othersubstitutions disclosed in the prior art.

Among said methods, preference is given according to the above tomethods, wherein the point mutations are as follows: 5A, 167R, 170P,177L, 202L, 204V, 271 D, 330D, 377R, 385S and/or 445Q.

The present invention further relates to nucleic acids coding for any ofthe above-described α-amylase variants according to the invention.

Said nucleic acids are understood as meaning both RNA and DNA moleculesand also DNA analogs, both the coding and the codogenic strand, that isin every reading frame, because it is possible, for example, to utilizethe teaching associated with the invention, in order to regulatecorresponding α-amylases via an interfering corresponding RNA or toincrease the lifetime of said genetic information by converting it to aDNA analog which is more slowly degradable in vivo. To a certain extent,said nucleic acids present the molecular-biological dimension to thepresent invention, as is discernible from the subject matters of theinvention set forth hereinbelow. According to the above, they arepreferred accordingly.

Said nucleic acids are understood as meaning preferably DNA moleculesbecause they can be used for preparing and/or, where appropriate,further modifying the α-amylase variants of the invention bymolecular-biological methods known per se. The nucleotide sequences ofthe abovementioned α-amylases established in the prior art are depositedin well-known databases (for example Genbank or Swissport; see above) orare revealed in said publications. These sequences are accordinglypreferred starting points for introducing point mutations according tothe invention.

Starting points for said nucleic acid may also be the sequenceinformation indicated in the sequences, in particular for derivatives ofBacillus sp. A 7-7 (DSM 12368) α-amylase. Another alternative consistsof preparing total DNA of particular microorganisms considered for thisand isolating the endogenous α-amylase genes by PCR. Primers which maybe employed in principle are the 5′ and 3′ sequence sections which canlikewise be read from SEQ ID NO. 1.

The pure protein-encoding sections may, for example, also be mutagenizedby a PCR, i.e. their sequence may be modified according to theinvention. To this end, a PCR with a corresponding statistical errorrate is carried out, the PCR product is cloned and the introducedmutations are verified by subsequent sequencing.

Preference is given here to a nucleic acid which derives from a nucleicacid according to SEQ ID NO. 1 or from a variant thereof havingincreasingly preferred 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 additionalpoint mutations to give those mutations resulting in any of the aminoacid substitutions according to the invention as defined above, by whichare meant those mutations by which at least one amino acid residue onthe surface of the starting molecule, which makes a neutral orpositively polar or charged contribution to the electrostatic potentialof said molecule has been replaced to give a more negatively polar ornegatively charged amino acid residue, meaning the stated ranking of theallowed amino acid substitutions or the leveling substitutions describedat from at least one to no more than exactly as many other positions ofone or more other amino acid residues on the surface of the startingmolecule to give less negatively polar or negatively charged amino acidresidues, meaning the correspondingly opposite ranking of the allowedamino acid substitutions.

This is because, as explained above, various point mutations can beintroduced into α-amylase molecules, which can exert advantageousactions in principle independently of one another. Thus it is possibleto carry out substitutions according to the invention on those moleculeswhich have been improved in particular with respect to specialperformance aspects, stability or, for example, allergenicity. Theyinclude in particular also non-inventive point mutations. This approachof generating in the end amino acid substitutions according to theinvention is based on the corresponding nucleic acids, and the order inwhich these various mutations are carried out is in principletechnically irrelevant here.

The present invention further relates to vectors which comprise apreviously specified nucleic acid of the invention.

This is because, in order to handle the nucleic acids relevant to theinvention and to carry out in particular the methods of the inventiontherewith and to prepare the production of proteins of the invention,they are suitably ligated into vectors. Such vectors and thecorresponding methods are described in detail in the prior art. A largenumber and variety of vectors are commercially available, both forcloning and for expression. These include, for example, vectors derivedfrom bacterial plasmids, from bacteriophages or from viruses, or mainlysynthetic vectors. They are further distinguished by the nature of thecell types in which they are able to establish themselves, for exampleaccording to vectors for gram-negative, gram-positive bacteria, foryeasts or for higher eukaryotes. They are suitable starting points, forexample for molecular-biological and biochemical studies, formutagenesis of the nucleic acid sections present therein and forexpression of the gene in question or the corresponding protein. Theyare virtually indispensable, in particular for preparing constructs fordeleting or enhancing expression, as is revealed by the relevant priorart.

Vectors are particularly suitable for making sequence variationsaccording to the invention, for example via site-directed mutagenesis,as illustrated in Example 3.

In one embodiment, vectors according to the invention are cloningvectors.

This is because cloning vectors are suitable for molecular-biologicalcharacterization of the gene of interest, in addition to its storage,biological amplification or selection. At the same time, they aretransportable and storable forms of the claimed nucleic acids and arealso starting points for molecular-biological techniques not associatedwith cells, such as, for example, PCR or in vitro mutagenesis methods.

It is possible, for example, to convert a cloning vector carrying thegene for an α-amylase described in the prior art to a cloning vector ofthe invention by carrying out a mutation according to the invention.

Vectors according to the invention are preferably expression vectors.

This is because such expression vectors are the basis of establishingthe corresponding nucleic acids in biological production systems andthereby producing the corresponding proteins, since they enable the geneproduct in question to be transcribed and translated, i.e. synthesized,in vivo. Preferred embodiments of this subject matter of the inventionare expression vectors that carry the genetic elements required forexpression, for example the natural promoter originally located upstreamof said gene or a promoter from a different organism. These elements maybe arranged, for example, in the form of an “expression cassette”.Alternatively, individual or all regulatory elements may also beprovided by the particular host cell. Particular preference is given toexpression vectors tuned to the chosen expression system, in particularthe host cell (see below), with respect to further properties, such as,for example, optimal copy number.

Providing an expression vector is usually the best option for amplifyinga protein according to the invention, preferably in combination withcoacting proteins according to the invention, and thus increasing theactivity in question or making it available to quantitative preparation.

A separate subject matter of the invention are cells which, aftergenetic modification, comprise any of the previously specified nucleicacids of the invention.

This is because said cells contain genetic information for the synthesisof a protein according to the invention and are used in the method ofthe invention, if the α-amylases in question are obtainedbiotechnologically. Said cells are understood as meaning in particularthose cells which have been provided with the nucleic acids of theinvention by methods known per se or which derived from such cells. Thisinvolves selecting suitably those host cells which can be cultured in acomparatively simple manner and/or deliver high yields of product.

They enable, for example, the corresponding genes to be amplified, orelse mutagenesis thereof, if an in vivo metagenesis method is carriedout, or transcription and translation, and ultimately biotechnologicalproduction of the proteins in question. Said genetic information may bepresent either extrachromosomally as a separate genetic element, i.e.located on a plasmid in the case of bacteria, or integrated in achromosome. The choice of a suitable system depends on problems such as,for example, type and duration of storage of said gene, or of theorganism or the type of mutagenesis or selection. Such possibleimplementations are known to the molecular biologist.

This also explains the preferred embodiment, according to which saidnucleic acid in such a cell is part of a vector, in particular part ofan above-described vector of the invention.

This is because the above-described advantages in handling, storage,expression etc., of the nucleic acid in question are associatedtherewith.

Among these cells, preference is in each case given to a host cell thatis a bacterium, in particular one that secretes the α-amylase variantformed into the surrounding medium.

This is because bacteria are characterized by short generation times andlow demands on culturing conditions. This enables inexpensive methods tobe established. Moreover, there is plenty of variable experienceregarding bacteria in fermentation technology. gram-negative orgram-positive bacteria may be suitable for a special production for verydifferent reasons which may be determined experimentally in theindividual case, such as nutrient sources, rate of product formation,time required, etc.

An additional advantageous embodiment relates to the fact that mostindustrially important α-amylases have originally been found as enzymessecreted by the relevant microorganisms, in particular bacteria, intothe surrounding medium. This is because they are usually digestiveenzymes in vivo. Accordingly, it is advantageous if the industriallyproduced enzymes of the invention are likewise secreted into thesurrounding medium, since they can be worked up therefrom without celldisruption and therefore comparatively easily. This may be achieved, forexample, by adding appropriate sequences to the genes in question—ifthese are not present anyway—that code for a leader peptide which causesthe cell in question to export them. An alternative in gram-negativebacteria, for example, consists of partially opening the outer membraneto release proteins, as described, for example, in the application WO01/81597 A1.

In a preferred embodiment, the bacterium is a gram-negative bacterium,in particular one of the genera Escherichia coli, Klebsiella,Pseudomonas or Xanthomonas, in particular E. coli K12, E. coli B orKlebsiella planticola strains, and very particular derivatives of thestrains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5a, E.coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

This is because these species and strains are established in the priorart for molecular-biological procedures and biotechnological productionprocesses. They are moreover available via generally accessible straincollections such as the Deutsche Sammlung von Mikroorganismen andZellkulturen gmbH, Mascheroder Web 1 b, 38124 Braunschweig, Germany orfrom commercial sources. In addition, they can be optimized for specificproduction conditions in combination with other components such as, forexample, vectors, which are likewise available in large numbers.

Gram-negative bacteria such as, for example, E. coli secrete amultiplicity of proteins into the periplasmic space. This may beadvantageous for special applications. There are, as mentioned, alsoprocesses known, by which gram-negative bacteria too export theexpressed proteins.

In an alternative embodiment which is no less preferred, the bacteriumis a gram-positive bacterium, in particular one of the genera Bacillus,Staphylococcus or Corynebacterium, very particularly of the speciesBacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B.globigii or B. alcalophilus, Staphylococcus carnosus or Corynebacteriumglutamicum, and among these in turn, very particularly preferably a B.licheniformis DSM 13 derivative.

This is because gram-positive bacteria are fundamentally different fromgram-negative ones in that secreted proteins are immediately releasedinto the nutrient medium surrounding the cells, from which medium theexpressed proteins according to the invention can be purified directly,if desired. Moreover, they are related to most source organisms forindustrially important enzymes or are identical thereto and usuallyproduce themselves comparable enzymes so that they have a similar codonusage and their protein synthesis apparatus is by nature geared theretoaccordingly. B. licheniformis DSM 13 which characterizes a veryparticularly preferred embodiment is likewise a very commonbiotechnological producer strain in the prior art.

Another embodiment of the present invention consists of methods ofproducing an above-described α-amylase variant.

The, in a narrower sense inventive, methods of increasing the stabilityof an α-amylase to multimerization brought about by electrostaticinteractions have already been described above. They refer primarily tomolecular-biological steps in order to generate such variants in thefirst place. The methods defined at this point, which are in a widersense likewise inventive, are those which are industrially required inorder to produce quantitative amounts of the α-amylase variants of theinvention. Thus they primarily refer to biotechnological methods ofproducing variants according to the invention—apart from the rather onlytheoretically relevant chemical synthesis of said enzymes. Said methodsusually involve microorganism strains which, by applyingmolecular-biological techniques known per se, have been enabled toproduce α-amylase variants which in the course of the present inventionhave been acknowledged as being advantageous according to the invention.

The method of the invention, which has been illustrated and which isbased in particular on mutagenesis, may thus be introduced as acharacterizing section into an established biotechnological method ofproducing α-amylases, i.e. may be combined with such a method.

The formation of a protein in question in a strain employed forproduction is detected by way of enzymic detection of the enzymeactivities in question by detection reactions known per se for α-amylaseactivity. For detection at the molecular-biological level, the proteinsdepicted in the present sequence listing can be synthesized by customarymethods and antibodies can be raised thereto. These proteins can then bedetected, for example, via appropriate Western blots. Said antibodiesreact particularly preferably to those surface regions which have beenmodified according to the invention, since the latter can bedistinguished from the non-inventive variants.

The aim of these methods is to deliver the α-amylases in question totheir particular applications. To this end, any work-up, purificationand formulation steps established in biotechnology can be used. Theseinclude, for example, the method of refining concentrated enzymesolutions, described in the application WO 2004/067557 A1, which ischromatography-based and which consequently can also be appliedsuccessfully to amylase preparations. Said method is the basis of theapplication DE 10360841.9 which has not been prepublished and accordingto which the solutions in question which have been obtained inter aliaby a chromatographic step can be processed further to give enzymegranules. Further method aspects which may likewise be combinedadvantageously with the present invention are revealed in theapplications DE 102004021384.4 and DE 192004019528 which have likewisenot been prepublished and according to which the storage stability andabrasion resistance of such granules can be increased by incorporatingglycerol or 1,2-propanediol or by a special coating.

Said methods, in particular their component steps, in which the enzymepreparation is still in a liquid form, are improved according to theinvention by the fact that the present α-amylase variant according tothe invention is less prone to aggregation via electrostaticinteractions, resulting in a smaller loss of total activity. Thisenables, for example, α-amylase granules to be produced which contain ahigher proportion of active substances.

The production methods are preferably methods which are carried outusing an above-specified nucleic acid of the invention, preferably usinga previously specified vector of the invention and particularlypreferably using a previously specified cell of the invention.

This is because said nucleic acids, in particular the nucleic acidsspecified in the sequence listing, make available the correspondinglypreferred genetic information in a microbiologically usable form, i.e.genetic engineering production processes. Increasing preference is givento providing said nucleic acids on a vector which can be utilizedparticularly successfully by the host cell, or such cells themselves.The production processes in question are known per se to the skilledworker.

Embodiments of the present invention may also be, on the basis of thecorresponding nucleic acid sequences, cell-free expression systems whichcarry out protein biosynthesis in vitro. All elements alreadyillustrated above may also be combined to give new methods for producingproteins according to the invention. In this connection, a multiplicityof possible combinations of method steps is conceivable for each proteinaccording to the invention and, as a result, optimal methods must bedetermined experimentally for each specific individual case.

Further preference is given to those methods in which one, preferablytwo or more and particularly preferably all codons of the nucleotidesequence have been adapted to the codon usage of the host strain. Thisis because the transfer of any of said genes to a less related speciesmay be utilized particularly successfully for the synthesis of theprotein in question, if their codon usage has been optimizedaccordingly.

Agents containing an above-described α-amylase variant of the inventionconstitute a separate subject-matter of the invention.

This means any agent in which the α-amylase in question is applied inany form, i.e. brought into the desired hydrolytic contact with itssubstrate, starch or starch-like polysaccharide, or is preparedtherefor. The desired contact usually takes place in an aqueousenvironment which is advantageously buffered to an appropriate pH and,where appropriate, contains further beneficial factors. These include,for example, further enzymes which further convert the immediatereaction products, for example with regard to starch liquefaction forthe production of food or animal feed or for ethanol production. Alsoincluded here are low-molecular weight compounds which are included, forexample, in nascent oligosaccharides, such as cyclodextrins, orlow-molecular compounds which further solubilize the cleavage productsor have a beneficial effect on the overall process, such as, forexample, surfactants within the framework of a detergent formulation.They may also be agents in which the desired analytical activity is tobe induced only with a large time delay, such as, for example, intemporary bonding processes, according to which the α-amylase variant isadded early to the adhesive in question but becomes actually active onlyafter a long time, due to an increase in the water content. Thisapplies, for example, also to detergents and cleansers which areintended to act on the substrate at the moment of dilution in the washliquor, only after a storage phase.

One embodiment of this subject matter of the invention are those agentswhich are a detergent or cleanser.

The properties and important ingredients according to the invention ofsuch detergents and cleansers are illustrated in more detailhereinbelow. At this point, there will be first an overview of the mostimportant embodiments, which are therefore particularly preferredaccording to the invention, of such detergents and cleansers:

-   -   A detergent or cleanser according to the invention, comprising        from 0.000001 percent by weight to 5% by weight, in particular        from 0.00001 to 3% by weight, of the α-amylase variant;    -   a detergent or cleanser according to the invention, which        additionally includes other enzymes, in particular hydrolytic        enzymes or oxidoreductases, particularly preferably further        amylases, proteases, lipases, cutinases, hemicellulases,        cellulases, β-glucanases, oxidases, peroxidases, perhydrolases        and/or laccases;    -   a detergent or cleanser according to the invention, wherein the        α-amylase variant is stabilized and/or its contribution to the        washing or cleaning performance of the agent is increased by any        of the other components of said agent;    -   a detergent or cleanser according to the invention, which is        overall solid, preferably after a compacting step for at least        one of the included components, particularly preferably that it        is overall compacted;    -   a detergent or cleanser according to the invention, which is        overall liquid, gel-like or paste-like, preferably with        encapsulation of at least one of the included components,        particularly preferably with encapsulation of at least one of        the included enzymes, very particularly preferably with        encapsulation of the α-amylase variant.

One important field of use of amylases is that as active components indetergents or cleansers for cleaning textiles or solid surfaces, suchas, for example, crockery, floors or utensils. In these applications,the amylolytic activity serves to break down by hydrolysis, or detachfrom the substrate, carbohydrate-containing contaminations and inparticular those based on starch. In this connection, they may be usedalone, in suitable media or else in detergents or cleansers. Theconditions to be chosen for this, such as, for example, temperature, pH,ionic strength, redox conditions or mechanical effects, should beoptimized for the particular cleaning problem, i.e. in relation to thesoiling and the substrate. Thus, usual temperatures for detergents andcleansers are in ranges from 10° C. for manual compositions via 40° C.and 60° C. up to 950 for machine compositions or for industrialapplications. Since the temperature can usually be adjusted continuouslyin modern washing and dishwashing machines, all intermediatetemperatures are also included. The ingredients of the relevant agentsare preferably also matched to one another. The other conditions canlikewise be designed very specifically for the particular cleaningpurpose via the other components of said agents, illustrated below.

Preferred detergents and cleansers of the invention are distinguished bythe washing or cleaning performance of the agent in question beingimproved by adding an α-amylase variant of the invention, compared withthe formulation without this variant, under any of the conditionsdefinable in this way. Preference is given to synergies with respect tocleaning performance.

An α-amylase variant of the invention can be used both in compositionsfor large-scale consumers or industrial users and in products for theprivate consumer, and all presentations which are expedient and/orestablished in the art also represent embodiments of the presentinvention. The detergents or cleansers of the invention thus mean anyconceivable types of cleaning compositions, both concentrates andcompositions to be applied in an undiluted form; for use on a commercialscale, in the washing machine or for washing or cleaning by hand. Theyinclude, for example, detergents for textiles, carpets or naturalfibers, for which agents the term detergent is used according to thepresent invention. They include also, for example, dishwashing agentsfor dishwashers or manual washing-up liquids or cleaners for hardsurfaces such as metal, glass, porcelain, ceramics, tiles, stone,painted surfaces, plastics, wood or leather; for these, the termcleanser is used according to the present invention. Any type ofdetergent or cleanser is an embodiment of the present invention, if anamylase of the invention has been added to it.

Embodiments of the present invention comprise any presentationsestablished in the art and/or any expedient presentations of thecompositions of the invention. These include, for example, solids,pulverulent, liquid, gel-like or paste-like compositions, whereappropriate also composed of two or more phases, compressed oruncompressed; they also include for example: extrudates, granules,tablets or pouches, packaged both in large containers and in portions.

α-Amylases are combined in agents of the invention, for example, withone or more of the following ingredients: nonionic, anionic and/orcationic surfactants, bleaches, bleach activators, bleach catalysts,builders and/or cobuilders, solvents, thickeners, sequestering agents,electrolytes, optical brighteners, antiredeposition agents, corrosioninhibitors, in particular silver protectants, soil release agents, colortransfer inhibitors, foam inhibitors, abrasives, dyes, fragrances,antimicrobial agents, UV stabilizers, enzymes such as, for example,proteases, (where appropriate other) amylases, lipases, cellulases,hemicellulases or oxidases, stabilizers, in particular enzymestabilizers, and other components known in the art.

The nonionic surfactants used are preferably alkoxylated, advantageouslyethoxylated, in particular primary alcohols having preferably from 8 to18 carbon atoms and, on average, from 1 to 12 mol of ethylene oxide (EO)per mole of alcohol, in which the alcohol radical can be linear or,preferably, methyl-branched in the 2-position or can comprise linear andmethyl-branched radicals in a mixture as are customarily present in oxoalcohol radicals. Particular preference is, however, given to alcoholethoxylates containing linear radicals of alcohols of native originhaving from 12 to 18 carbon atoms, for example from coconut, palm,tallow fatty or oleyl alcohol, and, on average, from 2 to 8 EO per moleof alcohol. Preferred ethoxylated alcohols include, for example,C₁₂₋₁₄-alcohols having 3 EO or 4 EO, C₉₋₁₁-alcohol having 7 EO,C₁₃₋₁₅-alcohols having 3 EO, 5 EO, 7 EO or 8 EO, C₁₂₋₁₈-alcohols having3 EO, 5 EO or 7 EO, and mixtures of these, such as mixtures ofC₁₂₋₁₄-alcohol having 3 EO and C₁₂₋₁₈-alcohol having 5 EO. The degreesof ethoxylation given are statistical averages which may be an integeror a fraction for a specific product. Preferred alcohol ethoxylates havea narrowed homolog distribution (narrow range ethoxylates, NRE). Inaddition to these nonionic surfactants, fatty alcohols having more than12 EO can also be used. Examples thereof are tallow fatty alcohol having14 EO, 25 EO, 30 EO or 40 EO.

A further class of preferably used nonionic surfactants which are usedeither as the sole nonionic surfactant or in combination with othernonionic surfactants are alkoxylated preferably ethoxylated orethoxylated and propoxylated fatty acid alkyl esters, preferably havingfrom 1 to 4 carbon atoms in the alkyl chain, in particular fatty acidmethyl esters.

The application DE 1020040430.6 which has not been published previouslyreveals cleansers, in particular machine dishwashing agents, which (a)comprise one or more nonionic surfactants having the following generalformula:

where R¹ is a C₆₋₂₄-alkyl or -alkenyl radical, the groups R² and R³ arein each case particular hydrocarbon radicals and the indices w, x, y, zare in each case integers up to 6, or a surfactant system of at leastone nonionic surfactant F of the general formula:

R¹—CH(OH)CH₂O-(AO)_(w)-(A′O)_(x—)(A″O)_(y)-(A′″O)_(z)—R²

and at least one nonionic surfactant g of the general formula:

R¹—O-(AO)_(w)-(A′O)_(x—)(A″O)_(y)-(A′″O)_(z)—R²

where in each case R¹ is a C₆₋₂₄-alkyl or -alkenyl radical, R² is ahydrocarbon radical having from 2 to 26 carbon atoms, A, A′, A″ and A′″are in each case particular hydrocarbon radicals, and w, x, y and z arein each case values of up to 25, said surfactant system having thesurfactants F and g in a weight ratio of between 1:4 and 100:1, and aspecial α-amylase variant as component (b).

These surfactants can be combined, in particular in machine dishwashingagents, also with α-amylases which correspond to the present invention,in particular if they have, besides the substitutions according to theinvention, those which can be found in one or more of the applicationsWO 96/23873 A1, WO 00/60060 A2 and WO 01/66712 A2. This applies inparticular to the case in which the commercial product Stainzyme® fromNovozymes, which falls under these applications, is improved in furtherpositions and is additionally provided with at least one substitution ofthe invention, since in principle an additive effect of the variousmodifications must be assumed.

A further class of nonionic surfactants which can advantageously be usedare the alkyl polyglycosides (APG). Alkyl polyglycosides which may beused satisfy the general formula RO(G)_(z), in which R is a linear orbranched, in particular methyl-branched in the 2-position, saturated orunsaturated, aliphatic radical having from 8 to 22, preferably from 12to 18 carbon atoms, and g is the symbol which stands for a glycose unithaving 5 or 6 carbon atoms, preferably for glucose. The degree ofglycosylation z is here between 1.0 and 4.0, preferably between 1.0 and2.0 and in particular between 1.1 and 1.4. Preference is given to usinglinear alkyl polyglucosides, i.e. alkyl polyglycosides in which thepolyglycosyl radical is a glucose radical, and the alkyl radical is ann-alkyl radical.

Nonionic surfactants of the amine oxide type, for exampleN-cocoalkyl-N,N-dimethylamine oxide and N-tallowalkyl-N,N-dihydroxyethylamine oxide, and of the fatty acid alkanolamidesmay also be suitable. The proportion of these nonionic surfactants ispreferably no more than that of the ethoxylated fatty alcohols, inparticular no more than half thereof.

Further suitable surfactants are polyhydroxy fatty acid amides of theformula (II)

in which RCO is an aliphatic acyl radical having from 6 to 22 carbonatoms, R¹ is hydrogen, an alkyl or hydroxyalkyl radical having from 1 to4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl radicalhaving from 3 to 10 carbon atoms and from 3 to 10 hydroxyl groups. Thepolyhydroxy fatty acid amides are known substances which can usually beobtained by reductive amination of a reducing sugar with ammonia, analkylamine or an alkanolamine and subsequent acylation with a fattyacid, a fatty acid alkyl ester or a fatty acid chloride.

The group of polyhydroxy fatty acid amides also includes compounds ofthe formula (III)

in which R is a linear or branched alkyl or alkenyl radical having from7 to 12 carbon atoms, R¹ is a linear, branched or cyclic alkyl radicalor an aryl radical having from 2 to 8 carbon atoms, and R² is a linear,branched or cyclic alkyl radical or an aryl radical or an oxy-alkylradical having from 1 to 8 carbon atoms, where C₁₋₄-alkyl or phenylradicals are preferred, and [Z] is a linear polyhydroxyalkyl radicalwhose alkyl chain is substituted with at least two hydroxyl groups, oralkoxylated, preferably ethoxylated or propoxylated, derivatives of thisradical.

[Z] is preferably obtained by reductive amination of a reducing sugar,for example glucose, fructose, maltose, lactose, galactose, mannose orxylose. The N-alkoxy- or N-aryloxy-substituted compounds may beconverted, for example by reaction with fatty acid methyl esters in thepresence of an alkoxide as catalyst, into the desired polyhydroxy fattyacid amides.

The anionic surfactants used are, for example, those of the sulfonateand sulfate type. Suitable surfactants of the sulfonate type arepreferably C₉₋₁₃-alkylbenzene sulfonates, olefin sulfonates, i.e.mixtures of alkene and hydroxyalkane sulfonates, and disulfonates, asobtained, for example, from C₁₂₋₁₈-monoolefins having a terminal orinternal double bond by sulfonation with gaseous sulfur trioxide andsubsequent alkaline or acidic hydrolysis of the sulfonation products.Also suitable are alkane sulfonates which are obtained fromC₁₂₋₁₈-alkanes, for example, by sulfochlorination or sulfoxidation withsubsequent hydrolysis or neutralization. Likewise suitable are also theesters of α-sulfo fatty acids (estersulfonates), for example theα-sulfonated methyl esters of hydrogenated coconut, palm kernel ortallow fatty acids.

Further suitable anionic surfactants are sulfated fatty acid glycerolesters. Fatty acid glycerol esters mean the mono-, di- and triesters,and mixtures thereof, as are obtained during the preparation byesterification of a monoglycerol with from 1 to 3 mol of fatty acid orduring the transesterification of triglycerides with from 0.3 to 2 molof glycerol. Preferred sulfated fatty acid glycerol esters are here thesulfation products of saturated fatty acids having from 6 to 22 carbonatoms, for example of caproic acid, caprylic acid, capric acid, myristicacid, lauric acid, palmitic acid, stearic acid or behenic acid.

Preferred alk(en)yl sulfates are the alkali metal, and in particular thesodium, salts of sulfuric half-esters of C₁₂-C₁₈-fatty alcohols, forexample of coconut fatty alcohol, tallow fatty alcohol, lauryl,myristyl, cetyl or stearyl alcohol or of C₁₀-C₂₀-oxo alcohols and thosehalf-esters of secondary alcohols of these chain lengths. Furtherpreferred are alk(en)yl sulfates of the said chain length which comprisea synthetic, petrochemical-based straight-chain alkyl radical which haveanalogous degradation behaviour to the equivalent compounds based onfatty chemical raw materials. From a washing performance viewpoint,preference is given to C₁₂-C₁₆-alkyl sulfates and C₁₂-C₁₅-alkylsulfates, and C₁₄-C₁₅-alkyl sulfates. 2,3-Alkyl sulfates are alsosuitable anionic surfactants.

The sulfuric monoesters of straight-chain or branched C₇₋₂₁-alcoholsethoxylated with from 1 to 6 mol of ethylene oxide, such as2-methyl-branched C₉₋₁₁-alcohols having, on average, 3.5 mol of ethyleneoxide (EO) or C₁₂₋₁₈-fatty alcohols having from 1 to 4 EO, are alsosuitable. Owing to their high foaming behaviour, they are used incleaning agents only in relatively small amounts, for example in amountsup to 5% by weight, usually from 1 to 5% by weight

Further suitable anionic surfactants are also the salts ofalkylsulfosuccinic acid, which are also referred to as sulfosuccinatesor as sulfosuccinic esters and which are monoesters and/or diesters ofsulfosuccinic acid with alcohols, preferably fatty alcohols and, inparticular, ethoxylated fatty alcohols. Preferred sulfosuccinatescontain C₈₋₁₈-fatty alcohol radicals or mixtures thereof. Particularlypreferred sulfosuccinates contain a fatty alcohol radical derived fromethoxylated fatty alcohols, which are themselves nonionic surfactants(see below for description). In this connection, sulfosuccinates whosefatty alcohol radicals are derived from ethoxylated fatty alcoholshaving a narrowed homologue distribution are, in turn, particularlypreferred. Likewise, it is also possible to use alk(en)ylsuccinic acidhaving preferably from 8 to 18 carbon atoms in the alk(en)yl chain orsalts thereof.

Further suitable anionic surfactants are, in particular, soaps.Saturated fatty acid soaps such as the salts of lauric acid, myristicacid, palmitic acid, stearic acid, hydrogenated erucic acid and behenicacid, and, in particular, soap mixtures derived from natural fattyacids, for example coconut, palm kernel or tallow fatty acids, aresuitable.

The anionic surfactants including soaps may be present in the form oftheir sodium, potassium or ammonium salts, and as soluble salts oforganic bases such as mono-, di- or triethanolamine. The anionicsurfactants are preferably in the form of their sodium or potassiumsalts, in particular in the form of the sodium salts.

The surfactants may be present in the cleaning agents or washing agentsaccording to the invention in an overall amount of from preferably 5% byweight to 50% by weight, in particular from 8% by weight to 30% byweight, based on the finished agent.

Agents according to the invention may contain bleaches. Of the compoundswhich serve as bleaches and produce H₂O₂ in water, sodium percarbonate,sodium perborate tetrahydrate and sodium perborate monohydrate are ofparticular importance. Other bleaches which can be used are, forexample, peroxopyrophosphates, citrate perhydrates and H₂O₂-producingperacidic salts or peracids, such as persulfates or persulfuric acid.Also useful is the urea peroxohydrate percarbamide which can bedescribed by the formula H₂N—CO—NH₂.H₂O₂. In particular when used forcleaning hard surfaces, for example for machine dishwashing, the agents,if desired, may also contain bleaches from the group of organicbleaches, although the use thereof is possible in principle also inagents for washing textiles. Typical organic bleaches are diacylperoxides such as, for example, dibenzoyl peroxide. Further typicalorganic bleaches are the peroxy acids, specific examples being alkylperoxy acids and aryl peroxy acids. Preferred representatives are peroxybenzoic acid and its ring-substituted derivatives, such asalkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesiummonoperphthalate, the aliphatic or substituted aliphatic peroxy acidssuch as peroxylauric acid, peroxystearic acid,∈-phthalimidoperoxycaproic acid (phthalimidoperoxyhexanoic acid, PAP),o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid andN-nonenylamidopersuccinate, and aliphatic and araliphaticperoxydicarboxylic acids such as 1,12-diperoxycarboxylic acid,1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid,diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-dioic acid,N,N-terephthaloyl-di(6-aminopercaproic acid) may be used.

The bleach content of the agents may be from 1 to 40% by weight and, inparticular, from 10 to 20% by weight, using advantageously perboratemonohydrate or percarbonate. The applications WO 99/63036 and WO99/63037, respectively, disclose a synergistic use of amylase withpercarbonate and of amylase with percarboxylic acid.

In order to achieve improved bleaching action in cases of washing attemperatures of 60° C. and below, and in particular in the case oflaundry pretreatment, the agents may also include bleach activators.Bleach activators which can be used are compounds which, underperhydrolysis conditions, give aliphatic peroxocarboxylic acids havingpreferably from 1 to 10 carbon atoms, in particular from 2 to 4 carbonatoms, and/or substituted or unsubstituted perbenzoic acid. Substanceswhich carry 0- and/or N-acyl groups of the said number of carbon atomsand/or substituted or unsubstituted benzoyl groups are suitable.Preference is given to plurally acylated alkylenediamines, in particulartetraacetylethylenediamine (TAED), acylated triazine derivatives, inparticular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT),acylated glycoluriles, in particular 1,3,4,6-tetraacetylglycolurile(TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI),acylated phenol sulfonates, in particular n-nonanoyl- orisononanoyloxybenzene sulfonate (n- or iso-NOBS), acylatedhydroxycarboxylic acids such as triethyl-O-acetyl citrate (TEOC),carboxylic anhydrides, in particular phthalic anhydride, isatoicanhydride and/or succinic anhydride, carboxamides such asN-methyldiacetamide, glycolide, acylated polyhydric alcohols, inparticular triacetin, ethylene glycol diacetate, isopropenyl acetate,2,5-diacetoxy-2,5-dihydrofuran and the enol esters disclosed in Germanpatent applications DE 196 16 693 and DE 196 16 767, and acetylatedsorbitol and mannitol, or mixtures thereof described in European patentapplication EP 0 525 239 (SORMAN), acylated sugar derivatives, inparticular pentaacetylglucose (PAG), pentaacetylfructose,tetraacetylxylose and octaacetyllactose, and acetylated, optionallyN-alkylated glucamine or gluconolactone, triazole or triazolederivatives and/or particulate caprolactams and/or caprolactamderivatives, preferably N-acylated lactams, for exampleN-benzoylcaprolactam and N-acetylcaprolactam. Hydrophilicallysubstituted acyl acetals and acyl lactams are likewise used withpreference. It is also possible to use combinations of conventionalbleach activators. Nitrile derivatives such as cyanopyridines, nitrilequats, e.g. N-alkylammoniumacetonitriles, and/or cyanamide derivativesmay also be used. Preferred bleach activators are sodium4-(octanoyloxy)benzenesulfonate, n-nonanoyl- orisononanoyloxybenzenesulfonate (n- or iso-NOBS),undecenoyloxybenzenesulfonate (UDOBS), sodiumdodecanoyloxybenzenesulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC10) and/or dodecanoyloxybenzenesulfonate (OBS12), andN-methylmorpholinium acetonitrile (MMA). Such bleach activators may bepresent in the customary quantitative range from 0.01 to 20% by weight,preferably in amounts from 0.1 to 15% by weight, in particular 1% byweight to 10% by weight, based on the total composition.

In addition to the conventional bleach activators or instead of them, itis also possible for “bleach catalysts” to be present. These substancesare bleach-enhancing transition metal salts or transition metalcomplexes such as, for example, Mn, Fe, Co, Ru or Mo saline complexes orcarbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexescontaining N-containing tripod ligands, and Co, Fe, Cu and Ru aminecomplexes are also suitable as bleach catalysts. Acetonitrilederivatives, according to WO 99/63038, and bleach-activating transitionmetal complex compounds, according to WO 99/63041 are capable ofdeveloping a bleach-activating action in combination with amylases.

Agents according to the invention usually contain one or more builders,in particular zeolites, silicates, carbonates, organic cobuilders and,where no ecological reasons oppose their use, also phosphates. Thelatter are the preferred builders for use in particular in cleaningagents for machine dishwashing.

Compounds which may be mentioned here are crystalline, layered sodiumsilicates of the general formula NaMSi_(x)O_(2x+1).yH₂O, where M issodium or hydrogen, x is a number from 1.6 to 4, preferably from 1.9 to4.0, and y is a number from 0 to 20, and preferred values for x are 2, 3or 4. Preferred crystalline phyllosilicates of the formula indicated arethose where M is sodium and x adopts the values 2 or 3. In particular,both β- and δ-sodium disilicates Na₂Si₂O₅.yH₂O are preferred. Compoundsof this kind are sold, for example, under the name SKS® (Clariant).Thus, SKS-6® is primarily a δ-sodium disilicate having the formulaNa₂Si₂O₅.yH₂O, and SKS-7® is primarily the β-sodium disilicate. Reactingthe δ-sodium disilicate with acids (for example citric acid or carbonicacid) gives kanemite NaHSi₂O₅.yH₂O, sold under the names SKS-9® and,respectively, SKS-10® (Clariant). It may also be advantageous to usechemical modifications of the said phyllosilicates. The alkalinity ofthe phyllosilicates, for example, can thus be suitably influenced.Phyllosilicates doped with phosphate or with carbonate have, compared tothe δ-sodium disilicate, altered crystal morphologies, dissolve morerapidly and display an increased calcium binding ability, compared toδ-sodium disilicate. Phyllosilicates of the general empirical formulaxNa₂O.ySiO₂.zP₂O₅ where the x-to-y ratio corresponds to a number from0.35 to 0.6, the x-to-z ratio to a number from 1.75 to 1200 and they-to-z ratio to a number from 4 to 2800 are to be mentioned inparticular. The solubility of the phyllosilicates may also be increasedby using particularly finely granulated phyllosilicates. It is alsopossible to use compounds of the crystalline phyllosilicates with otheringredients. Compounds which may be mentioned here are in particularthose with cellulose derivatives which have advantageous disintegratingaction and are used in particular in washing agent tablets, and thosewith polycarboxylates, for example citric acid, or polymericpolycarboxylates, for example copolymers of acrylic acid.

It is also possible to use amorphous sodium silicates having anNa₂O:SiO₂ modulus of from 1:2 to 1:3.3, preferably from 1:2 to 1:2.8,and in particular from 1:2 to 1:2.6, which have delayed dissolution andsecondary washing properties. The dissolution delay relative toconventional amorphous sodium silicates can have been induced by variousmeans, for example by surface treatment, compounding,compaction/compression or by overdrying. Within the scope of thisinvention, the term “amorphous” also means “X-ray amorphous”. This meansthat in X-ray diffraction experiments the silicates do not give thesharp X-ray refractions typical of crystalline substances, but instead,at best, one or more maxima of these scattered X-rays, which have awidth of several degree units of the diffraction angle. However,particularly good builder properties will very likely result if, inelectron diffraction experiments, the silicate particles give poorlydefined or even sharp diffraction maxima. This is to be interpreted tothe effect that the products have microcrystalline regions with a sizefrom 10 to a few hundred nm, preference being given to values up to atmost 50 nm and in particular up to at most 20 nm. Particular preferenceis given to compressed/compacted amorphous silicates, compoundedamorphous silicates and overdried X-ray amorphous silicates.

A finely crystalline, synthetic zeolite containing bonded water, whichmay be used where appropriate, is preferably zeolite A and/or P. Aszeolite P, zeolite MAP® (commercial product from Crosfield) isparticularly preferred. However, zeolite X and mixtures of A, X and/or Pare also suitable. A product which is commercially available and can beused with preference within the scope of the present invention is, forexample, also a co-crystallizate of zeolite X and zeolite A (approx. 80%by weight zeolite X), which is sold by CONDEA Augusta S.p.A. under thetrade name VEGOBOND AX® and can be described by the formula

nNa₂O.(1-n)K₂O.Al₂O₃.(2-2.5)SiO₂.(3.5-5.5)H₂O

Suitable zeolites have an average particle size of less than 10 μm(volume distribution; measurement method: Coulter counter) andpreferably contain from 18 to 22% by weight, in particular from 20 to22% by weight, of bonded water.

Use of the generally known phosphates as builder substances is of coursealso possible, provided such a use should not be avoided for ecologicalreasons. Among the multiplicity of commercially available phosphates,the alkali metal phosphates are the most important in the washing andcleaning agents industry, with pentasodium or pentapotassiumtriphosphate (sodium or potassium tripolyphosphate) being particularlypreferred.

In this connection, alkali metal phosphates is the collective term forthe alkali metal (in particular sodium and potassium) salts of thevarious phosphoric acids, it being possible to differentiate betweenmetaphosphoric acids (HPO₃)_(n) and orthophosphoric acid H₃PO₄ as wellas higher molecular weight representatives. The phosphates combineseveral advantages: they act as alkali carriers, prevent lime depositson machine parts and lime incrustations in fabrics and, moreover,contribute to the cleaning performance.

Sodium dihydrogenphosphate, NaH₂PO₄, exists as dihydrate (density 1.91gcm⁻³, melting point 60° C.) and as monohydrate (density 2.04 gcm⁻³).Both salts are white powders which are very readily soluble in water andwhich lose their water of crystallization upon heating and at 200° C.convert to the weakly acidic diphosphate (disodium hydrogendiphosphate,Na₂H₂P₂O₇), at a higher temperature to sodium trimetaphosphate (Na₃P₃O₉)and Maddrell's salt (see below). NaH₂PO₄ is acidic; it forms whenphosphoric acid is adjusted to a pH of 4.5 using sodium hydroxidesolution and the suspension is sprayed. Potassium dihydrogenphosphate(primary or monobasic potassium phosphate, potassium biphosphate, KDP),KH₂PO₄, is a white salt of density 2.33 gcm⁻³, has a melting point of253° [decomposition with the formation of potassium polyphosphate(KPO₃)_(x)] and is readily soluble in water.

Disodium hydrogenphosphate (secondary sodium phosphate), Na₂HPO₄, is acolorless crystalline salt which is very readily soluble in water. Itexists in anhydrous form and with 2 mol (density 2.066 gcm⁻³, loss ofwater at 95° C.), 7 mol (density 1.68 gcm⁻³, melting point 48° C. withloss of 5H₂O) and 12 mol (density 1.52 gcm⁻³, melting point 35° C. withloss of 5H₂O) of water, becomes anhydrous at 100° C. and upon morevigorous heating converts to the diphosphate Na₄P₂O₇. Disodiumhydrogenphosphate is prepared by neutralizing phosphoric acid with sodasolution using phenolphthalein as indicator. Dipotassiumhydrogenphosphate (secondary or dibasic potassium phosphate), K₂HPO₄, isan amorphous, white salt which is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na₃PO₄, are colorlesscrystals which, in the form of the dodecahydrate, have a density of 1.62gcm⁻³ and a melting point of 73-76° C. (decomposition), in the form ofthe decahydrate (corresponding to 19-20% P₂O₅) have a melting point of100° C. and in anhydrous form (corresponding to 39-40% P₂O₅) have adensity of 2.536 gcm⁻³. Trisodium phosphate is readily soluble in waterwith an alkaline reaction and is prepared by evaporating a solution ofexactly 1 mol of disodium phosphate and 1 mol of NaOH. Tripotassiumphosphate (tertiary or tribasic potassium phosphate), K₃PO₄, is a white,deliquescent granular powder of density 2.56 gcm⁻³, has a melting pointof 1340° C. and is readily soluble in water with an alkaline reaction.It is produced, for example, during the heating of Thomas slag withcarbon and potassium sulfate. Despite the higher price, the more readilysoluble, and therefore highly effective, potassium phosphates are oftenpreferred over corresponding sodium compounds in the cleaning agentsindustry.

Tetrasodium diphosphate (sodium pyrophosphate), Na₄P₂O₇, exists inanhydrous form (density 2.534 gcm⁻³, melting point 988° C., also 880° C.given) and as decahydrate (density 1.815-1.836 gcm⁻³, melting point 94°C. with loss of water). Both substances are colorless crystals whichdissolve in water with an alkaline reaction. Na₄P₂O₇ is formed duringthe heating of disodium phosphate to >200° C. or by reacting phosphoricacid with soda in a stoichiometric ratio and dewatering the solution byspraying. The decahydrate complexes heavy metal salts and hardnessconstituents and thus reduces the water hardness. Potassium diphosphate(potassium pyrophosphate), K₄P₂O₇, exists in the form of the trihydrateand is a colorless, hygroscopic powder of density 2.33 gcm⁻³, which issoluble in water, the pH of the 1% strength solution at 25° C. being10.4.

Condensation of NaH₂PO₄ and KH₂PO₄ results in higher molecular weightsodium phosphates and potassium phosphates, respectively, amongst whichcyclic representatives, the sodium and potassium metaphosphates,respectively, and chain-shaped types, the sodium and potassiumpolyphosphates, respectively, can be differentiated. Particularly forthe latter, a multiplicity of names are in use: melt or thermalphosphates, Graham's salt, Kurrol's and Maddrell's salt. All highersodium and potassium phosphates are together referred to as condensedphosphates.

The industrially important pentasodium triphosphate, Na₅P₃O₁₀ (sodiumtripolyphosphate), is a nonhygroscopic, white, water-soluble salt whichis anhydrous or crystallizes with 6H₂O and is of the general formulaNaO—[P(O)(ONa)—O]_(n)—Na where n=3. In 100 g of water, about 17 g of thesalt which is free of water of crystallization dissolve at roomtemperature, approx. 20 g dissolve at 60° C., and about 32 g dissolve at10° C.; if the solution is heated at 100° C. for two hours, about 8% oforthophosphate and 15% of diphosphate form due to hydrolysis. In thepreparation of pentasodium triphosphate, phosphoric acid is reacted withsoda solution or sodium hydroxide solution in a stoichiometric ratio,and the solution is dewatered by spraying. Similarly to Graham's saltand sodium diphosphate, pentasodium triphosphate dissolves manyinsoluble metal compounds (including lime soaps, etc.). Pentapotassiumtriphosphate, K₅P₃O₁₀ (potassium tripolyphosphate), is availablecommercially, for example, in the form of a 50% strength by weightsolution (>23% P₂O₅, 25% K₂O). The potassium polyphosphates are usedwidely in the washing and cleaning agents industry. In addition, sodiumpotassium tripolyphosphates also exist which can likewise be used withinthe scope of the present invention. These form, for example, when sodiumtrimetaphosphate is hydrolysed with KOH:

(NaPO₃)₃+2KOH→Na₃K₂P₃O₁₀+H₂O

According to the invention, these can be used exactly as sodiumtripolyphosphate, potassium tripolyphosphate or mixtures of these two;mixtures of sodium tripolyphosphate and sodium potassiumtripolyphosphate or mixtures of potassium tripolyphosphate and sodiumpotassium tripolyphosphate or mixtures of sodium tripolyphosphate andpotassium tripolyphosphate and sodium potassium tripolyphosphate canalso be used according to the invention.

Organic cobuilders which can be used in the washing and cleaning agentsaccording to the invention are, in particular, polycarboxylates orpolycarboxylic acids, polymeric polycarboxylates, polyaspartic acid,polyacetals, optionally oxidized dextrins, further organic cobuilders(see below), and phosphonates. These classes of substance are describedbelow.

Usable organic builder substances are, for example, the polycarboxylicacids usable in the form of their sodium salts, the term polycarboxylicacids meaning those carboxylic acids which carry more than one acidfunction. Examples of these are citric acid, adipic acid, succinic acid,glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid,sugar acids, aminocarboxylic acids, nitrilotriacetic acid (NTA), as longas such a use should not be avoided for ecological reasons, and mixturesthereof. Preferred salts are the salts of the polycarboxylic acids suchas citric acid, adipic acid, succinic acid, glutaric acid, tartaricacid, sugar acids, and mixtures thereof.

It is also possible to use the acids per se. In addition to theirbuilder action, the acids typically also have the property of anacidifying component and thus also serve to establish a lower and milderpH of washing or cleaning agents, as long as the pH resulting from themixture of the remaining components is not desired. Particular mentionshould be made here of system-compatible and environmentally safe acidssuch as citric acid, acetic acid, tartaric acid, malic acid, lacticacid, glycolic acid, succinic acid, glutaric acid, adipic acid, gluconicacid and any mixtures thereof. However, mineral acids, in particularsulfuric acid, or bases, in particular ammonium or alkali metalhydroxides, may also serve as pH regulators. The agents according to theinvention contain such regulators in amounts of preferably not more than20% by weight, in particular from 1.2% by weight to 17% by weight.

Suitable builders are also polymeric polycarboxylates; these are, forexample, the alkali metal salts of polyacrylic acid or ofpolymethacrylic acid, for example those having a relative molecular massof from 500 to 70 000 g/mol.

The molar masses given for polymeric polycarboxylates are, for thepurposes of this specification, weight-average molar masses, M_(W), ofthe respective acid form, determined in principle by means of gelpermeation chromatography (GPC), using a UV detector. The measurementwas made against an external polyacrylic acid standard which, owing toits structural similarity towards the polymers studied, providesrealistic molecular weight values. These figures differ considerablyfrom the molecular weight values obtained using polystyrenesulfonicacids as the standard. The molar masses measured againstpolystyrenesulfonic acids are usually considerably higher than the molarmasses given in this specification.

Suitable polymers are, in particular, polyacrylates which preferablyhave a molecular mass of from 2000 to 20 000 g/mol. Owing to theirsuperior solubility, preference in this group may be given in turn tothe short-chain polyacrylates which have molar masses of from 2000 to 10000 g/mol, and particularly preferably from 3000 to 5000 g/mol.

Also suitable are copolymeric polycarboxylates, in particular those ofacrylic acid with methacrylic acid and of acrylic acid or methacrylicacid with maleic acid. Copolymers which have proven to be particularlysuitable are those of acrylic acid with maleic acid which contain from50 to 90% by weight of acrylic acid and from 50 to 10% by weight ofmaleic acid. Their relative molecular mass, based on free acids, isgenerally from 2000 to 70 000 g/mol, preferably 20 000 to 50 000 g/moland in particular 30 000 to 40 000 g/mol. The (co)polymericpolycarboxylates may be used either as powders or as aqueous solution.The (co)polymeric polycarboxylates may be from 0.5 to 20% by weight, inparticular 1 to 10% by weight of the content of the agent.

To improve the solubility in water, the polymers may also containallylsulfonic acids such as, for example, allyloxybenzenesulfonic acidand methallylsulfonic acid as monomers.

Particular preference is also given to biodegradable polymers of morethan two different monomer units, for example those which contain, asmonomers, salts of acrylic acid and of maleic acid, and vinyl alcohol orvinyl alcohol derivatives, or those which contain, as monomers, salts ofacrylic acid and of 2-alkylallylsulfonic acid, and sugar derivatives.

Further preferred copolymers are those which preferably have, asmonomers, acrolein and acrylic acid/acrylic acid salts or acrolein andvinyl acetate.

Further preferred builder substances which may be mentioned are alsopolymeric aminodicarboxylic acids, their salts or their precursorsubstances. Particular preference is given to polyaspartic acids orsalts and derivatives thereof.

Further suitable builder substances are polyacetals which can beobtained by reacting dialdehydes with polyolcarboxylic acids having from5 to 7 carbon atoms and at least 3 hydroxyl groups. Preferredpolyacetals are obtained from dialdehydes such as glyoxal,glutaraldehyde, terephthalaldehyde and mixtures thereof and frompolyolcarboxylic acids such as gluconic acid and/or glucoheptonic acid.

Further suitable organic builder substances are dextrins, for exampleoligomers or polymers of carbohydrates, which can be obtained by partialhydrolysis of starches. The hydrolysis can be carried out by customaryprocesses, for example acid-catalyzed or enzyme-catalyzed processes. Thehydrolysis products preferably have average molar masses in the rangefrom 400 to 500 000 g/mol. Preference is given here to a polysaccharidehaving a dextrose equivalent (DE) in the range from 0.5 to 40, inparticular from 2 to 30, where DE is a common measure of the reducingaction of a polysaccharide compared with dextrose which has a DE of 100.It is possible to use both maltodextrins having a DE between 3 and 20and dried glucose syrups having a DE between 20 and 37, and also “yellowdextrins” and “white dextrins” with higher molar masses in the rangefrom 2000 to 30 000 g/mol.

The oxidized derivatives of such dextrins are their reaction productswith oxidation agents which are able to oxidize at least one alcoholfunction of the saccharide ring to the carboxylic acid function.Particularly preferred organic builders for agents according to theinvention are oxidized starches and derivatives thereof.

Oxydisuccinates and other derivatives of disuccinates, preferablyethylenediamine disuccinate, are also further suitable cobuilders. Here,ethylenediamine N,N′-disuccinate (EDDS) is preferably used in the formof its sodium or magnesium salts. In this connection, further preferenceis also given to glycerol disuccinates and glycerol trisuccinates.Suitable use amounts in zeolite-containing and/or silicate-containingformulations are between 3 and 15% by weight.

Further organic cobuilders which may be used are, for example,acetylated hydroxycarboxylic acids or salts thereof, which may also bepresent, where appropriate, in lactone form and which contain at least 4carbon atoms and at least one hydroxy group and at most two acid groups.

A further class of substance having cobuilder properties is thephosphonates. These are, in particular, hydroxyalkane and aminoalkanephosphonates. Among the hydroxyalkane phosphonates, 1-hydroxyethane1,1-diphosphonate (HEDP) is of particular importance as a cobuilder. Itis preferably used as sodium salt, the disodium salt being neutral andthe tetrasodium salt being alkaline (pH 9). Suitable aminoalkanephosphonates are preferably ethylenediaminetetramethylene phosphonate(EDTMP), diethylenetriaminepentamethylene phosphonate (DTPMP) and higherhomologues thereof. They are preferably used in the form of the neutralsodium salts, for example as the hexasodium salt of EDTMP or as thehepta- and octasodium salt of DTPMP. Here, preference is given to usingHEDP as builder from the class of phosphonates. In addition, theaminoalkane phosphonates have a marked heavy metal-binding capacity.Accordingly, particularly if the agents also contain bleaches, it may bepreferable to use aminoalkane phosphonates, in particular DTPMP, ormixtures of the said phosphonates.

In addition, all compounds which are able to form complexes withalkaline earth metal ions can be used as cobuilders.

The agents according to the invention may contain builder substances,where appropriate, in amounts of up to 90% by weight, and preferablycontain them in amounts of up to 75% by weight. Washing agents accordingto the invention have builder contents of, in particular, from 5% byweight to 50% by weight. In inventive agents for cleaning hard surfaces,in particular for machine cleaning of dishes, the builder substancecontent is in particular from 5% by weight to 88% by weight, withpreferably no water-insoluble builder materials being used in suchagents. A preferred embodiment of inventive agents for, in particular,machine cleaning of dishes contains from 20% by weight to 40% by weightwater-soluble organic builders, in particular alkali metal citrate, from5% by weight to 15% by weight alkali metal carbonate and from 20% byweight to 40% by weight alkali metal disilicate.

Solvents which may be used in the liquid to gelatinous compositions ofwashing and cleaning agents are, for example, from the group ofmonohydric or polyhydric alcohols, alkanolamines or glycol ethers, aslong as they are miscible with water in the given concentration range.Preferably, the solvents are selected from ethanol, n- or i-propanol,butanols, ethylene glycol methyl ether, ethylene glycol ethyl ether,ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether,diethylene glycol methyl ether, diethylene glycol ethyl ether, propyleneglycol methyl, ethyl or propyl ether, dipropylene glycol monomethyl ormonoethyl ether, diisopropylene glycol monomethyl or monoethyl ether,methoxy, ethoxy or butoxy triglycol, 1-butoxyethoxy-2-propanol,3-methyl-3-methoxybutanol, propylene glycol t-butyl ether, and mixturesof these solvents.

Solvents may be used in the liquid to gelatinous washing and cleaningagents according to the invention in amounts of between 0.1 and 20% byweight, but preferably below 15% by weight, and in particular below 10%by weight.

To adjust the viscosity, one or more thickeners or thickening systemsmay be added to compositions according to the invention. These highmolecular weight substances which are also called swell(ing) agentsusually soak up the liquids and swell in the process, convertingultimately into viscous true or colloidal solutions.

Suitable thickeners are inorganic or polymeric organic compounds.Inorganic thickeners include, for example, polysilicic acids, clayminerals, such as montmorillonites, zeolites, silicas and bentonites.The organic thickeners are from the groups of natural polymers, modifiednatural polymers and completely synthetic polymers. Such naturalpolymers are, for example, agar-agar, carrageen, tragacanth, gum arabic,alginates, pectins, polyoses, guar flour, carob seed flour, starch,dextrins, gelatins and casein. Modified natural substances which areused as thickeners are primarily from the group of modified starches andcelluloses. Examples which may be mentioned here arecarboxymethylcellulose and other cellulose ethers, hydroxyethylcelluloseand hydroxypropylcellulose, and carob flour ether. Completely syntheticthickeners are polymers such as polyacrylic and polymethacryliccompounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines,polyamides and polyurethanes.

The thickeners may be present in an amount up to 5% by weight,preferably from 0.05 to 2% by weight, and particularly preferably from0.1 to 1.5% by weight, based on the finished composition.

The washing and cleaning agent according to the invention may, whereappropriate, comprise, as further customary ingredients, sequesteringagents, electrolytes and further excipients.

The textile washing agents according to the invention may contain, asoptical brighteners, derivatives of diaminostilbenedisulfonic acid oralkali metal salts thereof. Suitable are, for example, salts of4,4′-bis(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)stilbene-2,2′-disulfonicacid or similarly constructed compounds which carry a diethanolaminogroup, a methylamino group, an anilino group or a 2-methoxyethylaminogroup instead of the morpholino group. In addition, brighteners of thesubstituted diphenylstyryl type may be present, for example the alkalimetal salts of 4,4′-bis(2-sulfostyryl)diphenyl,4,4′-bis(4-chloro-3-sulfostyryl)diphenyl, or4-(4-chlorostyryl)-4′-(2-sulfostyryl)diphenyl. Mixtures of theabovementioned optical brighteners may also be used.

Graying inhibitors have the function of keeping the soil detached fromthe textile fibre in suspension in the liquor. Suitable for this purposeare water-soluble colloids, usually organic in nature, for examplestarch, glue, gelatin, salts of ethercarboxylic acids or ethersulfonicacids of starch or of cellulose, or salts of acidic sulfuric esters ofcellulose or of starch. Water-soluble polyamides containing acidicgroups are also suitable for this purpose. Furthermore, starchderivatives other than those mentioned above may be used, for examplealdehyde starches. Preference is given to using cellulose ethers such ascarboxymethylcellulose (Na salt), methylcellulose, hydroxyalkylcelluloseand mixed ethers such as methylhydroxyethylcellulose,methylhydroxypropylcellulose, methylcarboxymethylcellulose, and mixturesthereof, for example in amounts of from 0.1 to 5% by weight, based onthe agents.

In order to protect against silver corrosion, silver corrosioninhibitors may be used in dishwashing cleaning agents according to theinvention. Such inhibitors are known in the prior art, for examplebenzotriazoles, iron(III) chloride or CoSO₄. As disclosed, silvercorrosion inhibitors which are particularly suitable for being usedtogether with enzymes are manganese, titanium, zirconium, hafnium,vanadium, cobalt, or cerium salts and/or complexes in which thespecified metals are present in any of the oxidation stages II, III, IV,V or VI. Examples of such compounds are MnSO₄, V₂O₅, V₂O₄, VO₂, TiOSO₄,K₂TiF₆, K₂ZrF₆, Co(NO₃)₂, Co(NO₃)₃, and mixtures thereof.

Soil-release active ingredients or soil repellents are usually polymerswhich, when used in a washing agent, impart soil-repellent properties tothe laundry fiber and/or assist the ability of the other washing agentingredients to detach soil. A comparable effect can also be observedwith their use in cleaning agents for hard surfaces.

Soil-release active ingredients which are particularly effective andhave been known for a long time are copolyesters having dicarboxylicacid, alkylene glycol and polyalkylene glycol units. Examples thereofare copolymers or mixed polymers of polyethylene terephthalate andpolyoxyethylene glycol and acidic agents containing, inter alia, acopolymer of a dibasic carboxylic acid and an alkylene or cycloalkylenepolyglycol. Polymers of ethylene terephthalate and polyethylene oxideterephthalate and also copolyesters of ethylene glycol, polyethyleneglycol, aromatic dicarboxylic acid and sulfonated aromatic dicarboxylicacid in particular molar ratios may also be used advantageously. Alsoknown are methyl or ethyl group end-group-capped polyesters havingethylene and/or propylene terephthalate and polyethylene oxideterephthalate units, and washing agents containing such a soil-releasepolymer, and also polyesters which contain, in addition to oxyethylenegroups and terephthalic acid units, also substituted ethylene units andglycerol units. Also known are polyesters which contain, in addition tooxyethylene groups and terephthalic acid units, 1,2-propylene,1,2-butylene and/or 3-methoxy-1,2-propylene groups, and glycerol unitsand which are end-group-capped with C₁- to C₄-alkyl groups, and alsopolyesters having polypropylene terephthalate and polyoxyethyleneterephthalate units, which are at least partially end-group-capped byC₁₋₄-alkyl or acyl radicals. Also known are sulfoethyl end-group-cappedterephthalate-containing soil-release polyesters, and soil-releasepolyesters having terephthalate, alkylene glycol and poly-C-glycolunits, produced by sulfonation of unsaturated end groups. Othersubstances which have been disclosed are acidic, aromatic polyesterscapable of detaching soil, and nonpolymeric soil-repellent activeingredients for materials made of cotton, which have a plurality offunctional units: a first unit which may be cationic, for example, isable to adsorb to the cotton surface by means of electrostaticinteraction, and a second unit which is hydrophobic is responsible forthe active ingredient remaining at the water/cotton interface.

The color transfer inhibitors suitable for use in textile washing agentsaccording to the invention include, in particular,polyvinylpyrrolidones, polyvinylimidazoles, polymeric N-oxides such aspoly(vinylpyridine N-oxide) and copolymers of vinylpyrrolidone withvinylimidazole.

For use in machine cleaning processes, it may be of advantage to addfoam inhibitors to the agents. Examples of suitable foam inhibitors aresoaps of natural or synthetic origin having a high proportion of C₁₈-C₂₄fatty acids. Examples of suitable nonsurfactant-type foam inhibitors areorganopolysiloxanes and their mixtures with microfine, optionallysilanized silica and also paraffins, waxes, microcrystalline waxes, andmixtures thereof with silanized silica or bis-stearyl-ethylenediamide.With advantages, use is also made of mixtures of different foaminhibitors, for example mixtures of silicones, paraffins or waxes. Thefoam inhibitors, in particular those containing silicone and/orparaffin, are preferably bound to a granular, water-soluble ordispersible support substance. Particular preference is given here tomixtures of paraffins and bis-stearylethylenediamides.

The application DE 102004020430.6 which has not been publishedpreviously reveals cleansers, in particular machine dishwashing agents,which contain a copolymer of (i) unsaturated carboxylic acids, (ii)monomers containing sulfonic acid groups and (iii) optionally furtherionic or nonionogenic monomers and a special α-amylase variant. Saidcopolymers can also be combined with α-amylases according to theinvention, in particular if the latter, in addition to the substitutionsaccording to the invention, have substitutions of the kind that can befound in one or more of the applications WO 96/23873 A1, WO 00/60060 A2and WO 01/66712 A2. This applies in particular if the commercial productStainzyme® from Novozymes, which falls under these applications, isimproved in further positions and is additionally provided with at leastone substitution of the invention. This is because an additive effect ofthe various modifications must be assumed in principle.

A cleaning agent according to the invention for hard surfaces may, inaddition, contain ingredients with abrasive action, in particular fromthe group comprising quartz flours, wood flours, polymer flours, chalksand glass microbeads, and mixtures thereof. Abrasives are present in thecleaning agents according to the invention preferably at not more than20% by weight, in particular from 5% by weight to 15% by weight.

Dyes and fragrances are added to washing and cleaning agents in order toimprove the aesthetic appeal of the products and to provide theconsumer, in addition to washing and cleaning performance, with avisually and sensorially “typical and unmistakable” product. As perfumeoils and/or fragrances it is possible to use individual odorantcompounds, for example the synthetic products of the ester, ether,aldehyde, ketone, alcohol and hydrocarbon types. Odorant compounds ofthe ester type are, for example, benzyl acetate, phenoxyethylisobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate,dimethylbenzylcarbinyl acetate, phenylethyl acetate, linalyl benzoate,benzyl formate, ethyl methylphenyl glycinate, allylcyclohexylpropionate, styrallyl propionate and benzyl salicylate. The ethersinclude, for example, benzyl ethyl ether; the aldehydes include, forexample, the linear alkanals having 8-18 carbon atoms, citral,citronellal, citronellyloxyacetaldehyde, cyclamenaldehyde,hydroxycitronellal, lilial and bourgeonal; the ketones include, forexample, the ionones, α-isomethylionone and methyl cedryl ketone; thealcohols include anethol, citronellol, eugenol, geraniol, linalool,phenylethyl alcohol, and terpineol; the hydrocarbons include primarilythe terpenes such as limonene and pinene. Preference, however, is givento the use of mixtures of different odorants which together produce anappealing fragrance note. Such perfume oils may also contain naturalodorant mixtures, as obtainable from plant sources, for example pineoil, citrus oil, jasmine oil, patchouli oil, rose oil or ylang-ylangoil. Likewise suitable are muscatel, sage oil, camomile oil, clove oil,balm oil, mint oil, cinnamon leaf oil, lime blossom oil, juniper berryoil, vetiver oil, olibanum oil, galbanum oil and labdanum oil, and alsoorange blossom oil, neroli oil, orangepeel oil and sandalwood oil. Thedye content of washing and cleaning agents is usually less than 0.01% byweight, while fragrances may make up to 2% by weight of the overallformulation.

The fragrances may be incorporated directly into the washing andcleaning agents; however, it may also be advantageous to apply thefragrances to carriers which intensify the adhesion of the perfume tothe material to be cleaned and, by means of slower fragrance release,ensure long-lasting fragrance, in particular of treated textiles.Materials which have become established as such carriers are, forexample, cyclodextrins, it being possible, in addition, for thecyclodextrin-perfume complexes to be additionally coated with furtherauxiliaries. Another preferred carrier for fragances is the describedzeolite X which can also absorb fragrances instead of or in a mixturewith surfactants. Preference is therefore given to washing and cleaningagents which contain the described zeolite X and fragrances which,preferably, are at least partially absorbed on the zeolite.

Preferred dyes whose selection is by no means difficult for the skilledworker have high storage stability and insensitivity to the otheringredients of the agents and to light, and also have no pronouncedaffinity for textile fibres, so as not to stain them.

To control microorganisms, washing or cleaning agents may containantimicrobial active ingredients. Depending on antimicrobial spectrumand mechanism of action, a distinction is made here betweenbacteriostatics and bactericides, fungistatics and fungicides, etc.Examples of important substances from these groups are benzalkoniumchlorides, alkylaryl sulfonates, halogen phenols and phenol mercuryacetate. The terms antimicrobial action and antimicrobial activeingredient have, within the teaching according to the invention, themeaning common in the art. Suitable antimicrobial active ingredients arepreferably selected from the groups of alcohols, amines, aldehydes,antimicrobial acids or their salts, carboxylic esters, acid amides,phenols, phenol derivatives, diphenyls, diphenylalkanes, ureaderivatives, oxygen acetals, nitrogen acetals and also oxygen andnitrogen formals, benzamidines, isothiazolines, phthalimide derivatives,pyridine derivatives, antimicrobial surfactant compounds, guanidines,antimicrobial amphoteric compounds, quinolines,1,2-dibromo-2,4-dicyanobutane, iodo-2-propylbutyl carbamate, iodine,iodophors, peroxo compounds, halogen compounds, and any mixtures of theabove.

The antimicrobial active ingredient may be selected from ethanol,n-propanol, isopropanol, 1,3-butanediol, phenoxyethanol, 1,2-propyleneglycol, glycerol, undecylenic acid, benzoic acid, salicylic acid,dihydracetic acid, o-phenylphenol, N-methylmorpholinoacetonitrile (MMA),2-benzyl-4-chlorophenol, 2,2′-methylenebis(6-bromo-4-chlorophenol),4,4′-dichloro-2′-hydroxydiphenyl ether (dichlosan),2,4,4′-trichloro-2′-hydroxydiphenyl ether (trichlosan), chlorohexidine,N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea,N,N′-(1,10-decanediyldi-1-pyridinyl-4-ylidene)-bis(1-octanamine)dihydrochloride,N,N′-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetra-azatetradecanediimidamide,glucoprotamines, antimicrobial surface-active quaternary compounds,guanidines including the bi- and polyguanidines, such as, for example,1,6-bis(2-ethylhexylbiguamidohexane)dihydrochloride,1,6-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)hexane tetrahydrochloride,1,6-di-(N₁,N₁′-phenyl-N₁,N₁-methyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-2,6-dichlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-N₁,N₁′-beta-(p-methoxyphenyl)diguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-alpha-methyl-beta-phenyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-p-nitrophenyldiguanido-N₅,N₅′)hexanedihydrochloride,omega:omega-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)-di-n-propyl etherdihydrochloride,omega:omega′-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)-di-n-propylether tetrahydrochloride,1,6-di-(N₁,N₁′-2,4-dichlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride, 1,6-di-(N₁,N₁′-p-methylphenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-2,4,5-trichlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride,1,6-di-N₁,N₁′-alpha-(p-chlorophenyl)ethyldiguanido-N₅,N₅′)hexanedihydrochloride,omega:omega-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)m-xylenedihydrochloride, 1,12-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)dodecanedihydrochloride, 1,10-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)decanetetrahydrochloride, 1,12-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)dodecanetetrahydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride, ethylene-bis(1-tolylbiguanide),ethylene-bis(p-tolylbiguanide),ethylene-bis(3,5-dimethylphenylbiguanide),ethylene-bis(p-tert-amylphenylbiguanide),ethylene-bis(nonylphenylbiguanide), ethylene-bis(phenylbiguanide),ethylene-bis(N-butylphenylbiguanide),ethylene-bis(2,5-diethoxyphenylbiguanide),ethylene-bis(2,4-dimethylphenylbiguanide),ethylene-bis(o-diphenylbiguanide), ethylene-bis(mixed amylnaphthylbiguanide), N-butylethylene-bis(phenylbiguanide),trimethylenebis(o-tolylbiguanide),N-butyl-trimethyl-bis(phenylbiguanide) and the corresponding salts suchas acetates, gluconates, hydrochlorides, hydrobromides, citrates,bisulfites, fluorides, polymaleates, N-cocoalkyl sarcosinates,phosphites, hypophosphites, perfluorooctanoates, silicates, sorbates,salicylates, maleates, tartrates, fumarates,ethylenediaminetetraacetates, iminodiacetates, cinnamates, thiocyanates,arginates, pyromellitates, tetracarboxybutyrates, benzoates, glutarates,monofluorophosphates, perfluoropropionates, and any mixtures thereof.Also suitable are halogenated xylene and cresol derivatives, such asp-chlorometacresol or p-chlorometaxylene, and natural antimicrobialactive ingredients of plant origin (for example from spices or herbs),animal origin and microbial origin. Preference may be given to usingantimicrobial surface-active quaternary compounds, a naturalantimicrobial active ingredient of plant origin and/or a naturalantimicrobial active ingredient of animal origin, most preferably atleast one natural antimicrobial active ingredient of plant origin fromthe group comprising caffeine, theobromine and theophylline andessential oils such as eugenol, thymol and geraniol, and/or at least onenatural antimicrobial active ingredient of animal origin from the groupcomprising enzymes such as milk protein, lysozyme and lactoperoxidase,and/or at least one antimicrobial surface-active quaternary compoundhaving an ammonium, sulfonium, phosphonium, iodonium or arsonium group,peroxo compounds and chlorine compounds. It is also possible to usesubstances of microbial origin, the “bacteriocines”.

The quaternary ammonium compounds (QACs) which are suitable asantimicrobial active ingredients have the general formula(R¹)(R²)(R³)(R⁴)N⁺X⁻ where R¹ to R⁴ are identical or differentC₁-C₂₂-alkyl radicals, C₇-C₂₈-aralkyl radicals or heterocyclic radicals,where two, or in the case of an aromatic incorporation as in pyridine,even three radicals, together with the nitrogen atom, form theheterocycle, for example a pyridinium or imidazolinium compound, and X⁻are halide ions, sulfate ions, hydroxide ions or similar anions. Foroptimal antimicrobial action, at least one of the radicals preferablyhas a chain length of from 8 to 18, in particular 12 to 16, carbonatoms.

QACs can be prepared by reacting tertiary amines with alkylating agentssuch as, for example, methyl chloride, benzyl chloride, dimethylsulfate, dodecyl bromide, or else ethylene oxide. The alkylation oftertiary amines having one long alkyl radical and two methyl groupsproceeds particularly readily, and the quaternization of tertiary amineshaving two long radicals and one methyl group can also be carried outwith the aid of methyl chloride under mild conditions. Amines which havethree long alkyl radicals or hydroxy-substituted alkyl radicals have lowreactivity and are preferably quaternized using dimethyl sulfate.

Examples of suitable QACs are benzalkonium chloride(N-alkyl-N,N-dimethylbenzylammonium chloride, CAS No. 8001-54-5),benzalkone B (m,p-dichlorobenzyldimethyl-C₁₋₂-alkylammonium chloride,CAS No. 58390-78-6), benzoxonium chloride(benzyldodecyl-bis(2-hydroxyethyl)ammonium chloride), cetrimoniumbromide (N-hexadecyl-N,N-trimethylammonium bromide, CAS No. 57-09-0),benzetonium chloride(N,N-dimethyl-N-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethyl]benzylammoniumchloride, CAS No. 121-54-0), dialkyldimethylammonium chlorides such asdi-n-decyldimethylammonium chloride (CAS No. 7173-51-5-5),didecyldimethylammonium bromide (CAS No. 2390-68-3),dioctyldimethylammonium chloride, 1-cetylpyridinium chloride (CAS No.123-03-5) and thiazoline iodide (CAS No. 15764-48-1), and mixturesthereof. Particularly preferred QACs are the benzalkonium chlorideshaving C₈-C₁₈-alkyl radicals, in particularC₁₂-C₁₄-alkylbenzyldimethylammonium chloride.

Benzalkonium halides and/or substituted benzalkonium halides arecommercially available, for example, as Barquat® ex Lonza, Marquat® exMason, Variquat® ex Witco/Sherex and Hyamine® ex Lonza, and Bardac® exLonza. Further commercially available antimicrobial active ingredientsare N-(3-chloroallyl)hexaminium chloride such as Dowicide® and Dowicil®ex Dow, benzethonium chloride such as Hyamine® 1622 ex Rohm & Haas,methylbenzethonium chloride such as Hyamine® 10× ex Rohm & Haas,cetylpyridinium chloride such as cepacol chloride ex Merrell Labs.

The antimicrobial active ingredients are used in amounts of from 0.0001%by weight to 1% by weight, preferably from 0.001% by weight to 0.8% byweight, particularly preferably from 0.005% by weight to 0.3% by weight,and in particular from 0.01 to 0.2% by weight.

The agents may contain UV absorbers which attach to the treated textilesand improve the light stability of the fibres and/or the light stabilityof other formulation constituents. UV absorbers mean organic substances(light protection filters) which are able to absorb ultravioletradiation and to emit the absorbed energy again in the form of radiationof longer wavelength, for example heat.

Compounds which have these desired properties are, for example, thecompounds which are active via radiationless deactivation andderivatives of benzophenone having substituents in position(s) 2 and/or4. Furthermore, also suitable are substituted benzotriazoles, acrylateswhich are phenyl-substituted in position 3 (cinnamic acid derivatives,with or without cyano groups in position 2), salicylates, organic Nicomplexes and natural substances such as umbelliferone and theendogenous urocanic acid. Of particular importance are biphenyl andespecially stilbene derivatives, for example those commerciallyavailable as Tinosorb® FD or Tinosorb® FR ex Ciba. UV-B absorbers whichmay be mentioned are: 3-benzylidenecamphor or 3-benzylidenenorcamphorand derivatives thereof, for example 3-(4-methylbenzylidene)camphor;4-aminobenzoic acid derivatives, preferably 2-ethylhexyl4-(dimethylamino)benzoate, 2-octyl 4-(dimethylamino)benzoate and amyl4-(dimethylamino)benzoate; esters of cinnamic acid, preferably2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl4-methoxycinnamate, 2-ethylhexyl 2-cyano-3,3-phenylcinnamate(octocrylenes); esters of salicylic acid, preferably 2-ethylhexylsalicylate, 4-isopropylbenzyl salicylate, homomethyl salicylate;derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2,2′-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid,preferably di-2-ethylhexyl 4-methoxybenzmalonate; triazine derivativessuch as, for example,2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine andoctyltriazone, or dioctylbutamidotriazones (Uvasorb® HEB);propane-1,3-diones such as, for example,1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione;ketotricyclo(5.2.1.0)decane derivatives. Further suitable are2-phenylbenzimidazole-5-sulfonic acid and its alkali metal, alkalineearth metal, ammonium, alkylammonium, alkanolammonium and glucammoniumsalts; sulfonic acid derivatives of benzophenones, preferably2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts; sulfonicacid derivatives of 3-benzylidenecamphor, such as, for example,4-(2-oxo-3-bornylidenemethyl)benzenesulfonic acid and2-methyl-5-(2-oxo-3-bornylidene)sulfonic acid and salts thereof.

Suitable typical UV-A filters are, in particular, derivatives ofbenzoylmethane, such as, for example,1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione,4-tert-butyl-4′-methoxydibenzoylmethane (Parsol 1789),1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione, and enamine compounds(BASF). The UV-A and UV-B filters may of course also be used inmixtures. In addition to the said soluble substances, insoluble lightprotection pigments, namely finely dispersed, preferably nanoized, metaloxides or salts, are also suitable for this purpose. Examples ofsuitable metal oxides are, in particular, zinc oxide and titaniumdioxide and also oxides of iron, zirconium, silicon, manganese,aluminium and cerium, and mixtures thereof. Salts which may be used aresilicates (talc), barium sulfate or zinc stearate. The oxides and saltsare already used in the form of the pigments for skin-care andskin-protective emulsions and decorative cosmetics. The particles hereshould have an average diameter of less than 100 nm, preferably between5 and 50 nm, and in particular between 15 and 30 nm. They can have aspherical shape, but it is also possible to use particles which have anellipsoidal shape or a shape deviating in some other way from thespherical form. The pigments may also be surface-treated, i.e.hydrophilicized or hydrophobicized. Typical examples are coated titaniumdioxides such as, for example, titanium dioxide T 805 (Degussa) orEusolex® T2000 (Merck); suitable hydrophobic coating agents are herepreferably silicones and, particularly preferably, trialkoxyoctylsilanesor simethicones. Preference is given to using micronized zinc oxide.

The UV absorbers are usually used in amounts of from 0.01% by weight to5% by weight, preferably from 0.03% by weight to 1% by weight.

To increase the washing or cleaning performance, agents according to theinvention may contain further enzymes in addition to the α-amylasevariants of the invention, wherein it is possible in principle to useany enzymes established for these purposes in the prior art. Theseinclude in particular proteases, further amylases, lipases,hemicellulases, cellulases or oxidoreductases, and preferably mixturesthereof. These enzymes are in principle of natural origin; starting fromthe natural molecules, improved variants for use in detergents andcleansers are available which are used with preference accordingly.Agents according to the invention preferably contain enzymes in totalamounts of from 1×10−8 to 5 percent by weight, based on active protein.The protein concentration may be determined with the aid of knownmethods, for example the BCA method (bicinchonic acid;2,2′-biquinolyl-4,4′-dicarboxylic acid) or the biuret method (A. G.Gornall, C. S. Bardawill and M. M. David, J. Biol. Chem., 177 (1948),pp. 751-766).

Among the proteases, preference is given to those of the subtilisintype. Examples thereof include the subtilisins BPN′ and Carlsberg,protease PB92, subtilisins 147 and 309, Bacillus lentus alkalineprotease, subtilisin DY and the enzymes thermitase, proteinase K and theproteases TW3 and TW7, all of which can be classified to the subtilasesbut no longer to the subtilisins in the narrower sense. SubtilisinCarlsberg is available in a developed form under the trade nameAlcalase® from Novozymes A/S, Bagsvaerd, Denmark. Subtilisins 147 and309 are sold under the trade names Esperase®, and Savinase®,respectively, from Novozymes. The variants listed under the name BLAP®are derived from the protease of Bacillus lentus DSM 5483 (WO 91/02792A1) and are described in particular in WO 92/21760 A1, WO 95/23221 A1,WO 02/088340 A2 and WO 03/038082 A2. The applications WO 03/054185 A1,WO 03/056017 A2, WO 03/055974 A2 and WO 03/054184 A1 reveal furtherusable proteases from various Bacillus sp. and B. gibsonii.

Further examples of useful proteases are the enzymes available under thetrade names Durazym®, Relase®, Everlase®, Nafizym®, Natalase®, Kannase®and Ovozymes® from Novozymes, those under the trade names Purafect®,Purafect® OxP and Properase® from Genencor, that under the trade nameProtosol® from Advanced Biochemicals Ltd, Thane, India, that under thetrade name Wuxi® from Wuxi Snyder Bioproducts Ltd., China, those underthe trade names Proleather® and Protease P® from Amano PharmaceuticalsLtd., Nagoya, Japan, and known as proteinase K-16 from Kao Corp., Tokyo,Japan.

Examples of amylases which may be employed additionally according to theinvention are the α-amylases of Bacillus licheniformis, of B.amyloliquefaciens or of B. stearothermophilus, and developments thereofwhich have been improved for use in detergents and cleansers. The B.licheniformis enzyme is available from Novozymes under the nameTermamyl® and from genencor under the name Purastar® ST. Developmentproducts of this α-amylase are available from Novozymes, under the namesDuramyl® and Termamyl® ultra, from genencor, under the name Purastar®OxAm, and from Daiwa Seiko Ink., Tokyo Japan, as Keistase®. The B.amyloliquefaciens α-amylase is sold by Novozymes under the name BAN®,and variants derived from the B. stearothermophilus α-amylase are soldunder the names BSG® and Novamyl®, likewise from Novozymes.

Enzymes which should additionally be emphasized for this purpose are theα-amylase from Bacillus sp. A 7-7 (DSM 12368) disclosed in theapplication WO 02/10356 A2 and the cyclodextrin glucanotransferase(CGTase) from B. agaradherens (DSM 9948), described in the applicationWO 02/44350. It is further possible to use the amylolytic enzymes whichbelong to the sequence space of α-amylases, which is defined in theapplication WO 03/002711 A2, and those described in the application WO03/054177 A2. It is also possible to use fusion products of themolecules mentioned, for example those of the application DE 10138753A1, or point mutations thereof.

Also suitable are the developments of α-amylase from Aspergillus nigerand A. oryzae, which are available under the trade name Fungamyl® fromNovozymes. Other examples of commercial products which may be used areAmylase-LT® and Stainzyme®, the latter of which likewise from Novozymes.

Agents of the invention may comprise lipases or cutinases, in particulardue to their triglyceride-cleaving activities, but also in order togenerate peracids in situ from suitable precursors. Examples thereofinclude the lipases which were originally derived from Humicolalanuginosa (Thermomyces lanufinosus) or have been developed, inparticular those having the D96L amino acid substitution. They are sold,for example, under the trade names Lipolase®, Lipolase® Ultra,LipoPrime®, Lipozyme® and Lipex® by Novozymes. It is furthermorepossible to use, for example, the cutinases which have originally beenisolated from Fusarium solani pisi and Humicola insolens. Lipases whichare also useful can be obtained under the names Lipase CE®, Lipase P®,Lipase B®, or Lipase CES®, Lipase AKG®, Bacillus sp. Lipase®, LipasaeAP®, Lipase M-AP® and Lipase AML® from AmaNO. Examples of lipases andcutinases from Genencor, which can be used, are those whose startingenzymes have originally been isolated from Pseudomonas mendocina andFusarium solanii. Other important commercial products which may bementioned are the M1 Lipase® and Lipomax® preparations originally soldby gist-Brocades and the enzymes sold under the names Lipase MY-30®,Lipase OF® and Lipase PL® by Meito Sangyo KK, Japan, and also theproduct Lumafast® from Genencor.

Agents according to the invention may, in particular when intended forthe treatment of textiles, comprise cellulases, depending on the purposeeither as pure enzymes, as enzyme preparations or in the form ofmixtures in which the individual components advantageously complementone another with respect to their different performance aspects. Theseperformance aspects include in particular contributions to the primarywashing performance, to the secondary washing performance of the agent(antiredeposition action or graying inhibition) and finishing (fabricaction), up to exerting a “stonewashed” effect.

A useful fungal, endoglucanase (EG)-rich cellulase preparation anddevelopments thereof are supplied under the tradename Celluzyme® byNovozymes. The products Endolase® and Carezyme®, likewise available fromNovozymes, are based on the H. insolens DSM 1800 50 kD EG and 43 kD EGrespectively. Further commercial products of this company, which may beused, are Cellusoft® and Renozyme®. The latter is based on theapplication WO 96/29397 A1. Performance enhanced cellulase variants canbe found, for example, in the application WO 98/12307 A1. It is alsopossible to use the cellulases disclosed in the application WO 97/14804A1; for example the Melanocarpus 20 kD EG disclosed therein, which isavailable from AB Enzymes, Finland, under the trade names Ecostone® andBiotouch®. Further commercial products from AB Enzymes are Econase® andEcopulp®. WO 96/34092 A2 discloses further suitable cellulases fromBacillus sp. CBS 670.93 and CBS 669.93, with that of Bacillus sp. CBS670.93 being available under the trade name Puradax® from Genencor.Other commercial products from Genencor are “Genencor detergentcellulase L” and IndiAge® Neutra.

Agents according to the invention may comprise further enzymes, inparticular for removing particular problematic soilings, which arecombined under the term hemicellulases. These include, for example,mannanases, xanthane lyases, pectin lyases (=pectinases), pectinesterases, pectate lyases, xyloglucanases (=xylanases), pullulanases andβ-glucanases. Examples of suitable mannanases are available under thenames Gamanase® and Pektinex AR® from Novozymes, under the name Rohapec®B1L from AB Enzymes, under the name Pyrolase® from Diversa Corp., SanDiego, Calif., USA, and under the name Purabrite® from Genencor Int.,Inc., Palo Alto, Calif., USA. An example of a suitable β-glucanase froma B. alcalophilus is revealed by the application WO 99/06573 A1. Theβ-glucanase obtained from B. subtilis is available under the nameCereflo® from Novozymes.

To enhance the bleaching action, detergents and cleansers according tothe invention may comprise oxidoreductases, for example oxidases,oxygenases, katalases, peroxidases such as haloperoxidases,chloroperoxidases, bromoperoxidases, lignin peroxidases, glucoseperoxidases or manganese peroxidases, dioxygenases or laccases (phenoloxidases, polyphenol oxidases). Suitable commercial products which maybe mentioned are Denilite® 1 and 2 from Novozymes. Reference may be madeto the application WO 98/45398 A1 with respect to examples of systemsfor an enzymic perhydrolysis, which may be used advantageously. WO2004/058955 A2, for example, discloses choline oxidases useful inparticular for such a system. The application WO 2004/058961 A1 revealsmodified proteases having a pronounced perhydrolase activity which maylikewise be used advantageously here, in particular for achieving mildbleaching in detergents for textiles. The application DE 102004029475.5describes a combined enzymic bleaching system comprising an oxidase anda perhydrolase. WO 2005/056782 A2 also discloses further perhydrolaseswhich may be used according to the invention. Advantageously, preferablyorganic, more preferably aromatic, compounds which interact with theenzymes are additionally added in order to enhance the activity of theoxidoreductases in question (enhancers), or to ensure the electron fluxin the event of large differences in the redox potentials of theoxidizing enzymes and the soilings (mediators).

The enzymes used in agents according to the invention either originatefrom microorganisms, for example of the genera Bacillus, Streptomyces,Humicola, or Pseudomonas, and/or are produced in biotechnology processesknown per se by suitable microorganisms, for example by transgenicexpression hosts of the Bacillus genera or by filamentous fungi.

The enzymes in question are favorably purified via processes which areestablished per se, for example via precipitation, sedimentation,concentration, filtration of the liquid phases, microfiltration,ultrafiltration, the action of chemicals, deodorization or suitablecombinations of these steps.

The enzymes may be added to the agents according to the invention in anyform established in the prior art, including, for example, the solidpreparations obtained by granulation, extrusion or lyophilization, or,in particular in the case of liquid or gel-like agents, solutions of theenzymes, advantageously highly concentrated, low in water and/or admixedwith stabilizers.

Alternatively, the enzymes may be encapsulated both for solid and liquidforms of presentation, for example by spray-drying or extrusion of theenzyme solution together with a, preferably natural, polymer, or in theform of capsules, for example those in which the enzymes are enclosed asin a solidified gel, or in those of the core-shell type, in which anenzyme-containing core is coated with a water-, air- and/orchemical-impermeable protective layer. It is possible to additionallyapply further active ingredients, for example stabilizers, emulsifiers,pigments, bleaches or dyes, in the form of layers applied thereupon.Such capsules are applied by methods known per se, for example byagitated or roll granulation or in fluidized bed processes.Advantageously, such granules are low-dusting, for example due toapplication of polymeric film formers, and storage-stable as a result ofsaid coating.

It is furthermore possible to formulate two or more enzymes together, sothat a single granule has a plurality of enzyme activities.

A protein and/or enzyme present in an agent of the invention may beprotected, particularly during storage, from damage such as, forexample, inactivation, denaturation or decay, for instance due tophysical influences, oxidation or proteolytic cleavage. When saidproteins and/or enzymes are produced microbially, particular preferenceis given to inhibiting proteolysis, in particular when the agents alsocomprise proteases. For this purpose, preferred agents according to theinvention comprise stabilizers.

One group of stabilizers are reversible protease inhibitors. Frequently,benzamidine hydrochloride, borax, boric acids, boronic acids or salts oresters thereof are used, and of these especially derivatives havingaromatic groups, for example ortho-, meta- or para-substitutedphenylboronic acids, in particular 4-formylphenylboronic acid, or thesalts or esters of said compounds. Peptide aldehydes, i.e. oligopeptideswith reduced C terminus, in particular those composed of from 2 to 50monomers, are also used for this purpose. Peptidic reversible proteaseinhibitors include inter alia ovomucoid and leupeptin. Specific,reversible peptide inhibitors of the protease subtilisin and also fusionproteins of proteases and specific peptide inhibitors are also suitablefor this.

Further enzyme stabilizers are amino alcohols such as mono-, di-,triethanol- and -propanolamine and mixtures thereof, aliphaticcarboxylic acids up to C₁₂, such as succinic acid for example, otherdicarboxylic acids or salts of said acids. End group-capped fatty acidamide alkoxylates are also suitable for this purpose. Particular organicacids used as builders are capable of additionally stabilizing an enzymepresent, as disclosed in WO 97/18287.

Lower aliphatic alcohols, but especially polyols such as, for example,glycerol, ethylene glycol, propylene glycol or sorbitol, are otherfrequently used enzyme stabilizers. Diglycerol phosphate also protectsagainst denaturation due to physical influences. Calcium salts and/ormagnesium salts are also used, for example calcium acetate or calciumformate.

Polyamide oligomers or polymeric compounds such as lignin, water-solublevinyl copolymers or cellulose ethers, acrylic polymers and/orpolyamides, stabilize the enzyme preparation inter alia to physicalinfluences or pH fluctuations. Polyamine N-oxide-containing polymers actsimultaneously as enzyme stabilizers and as color transfer inhibitors.Other polymeric stabilizers are the linear C₈-C₁₈ polyoxyalkylenes.Alkylpolyglycosides can likewise stabilize the enzymic components of theagent of the invention and are preferably able to increase in additionthe performance of said components. Crosslinked N-containing compoundsfulfill a double function as soil release agents and as enzymestabilizers. Hydrophobic, nonionic polymer stabilizes in particular anoptionally present cellulase.

Reducing agents and antioxidants increase the stability of the enzymesto oxidative decay; familiar examples thereof are sulfur-containingreducing agents. Other examples are sodium sulfite and reducing sugars.

Particular preference is given to using combinations of stabilizers, forexample of polyols, boric acid and/or borax, the combination of boricacid or borate, reducing salts and succinic acid or other dicarboxylicacids, or the combination of boric acid or borate with polyols orpolyamino compounds and with reducing salts. The action ofpeptide-aldehyde stabilizers can be increased advantageously bycombination with boric acid and/or boric acid derivatives and polyols,and furthermore by the additional action of divalent cations, forexample calcium ions.

In a preferred embodiment, agents according to the invention arecharacterized in that they are composed of more than one phase, forexample in order to release the active ingredients separately from oneanother with regard to time or space. Said phases may be in variousstates of aggregation but in particular in the same state ofaggregation.

Agents according to the invention which are composed of more than onesolid component may be prepared in a simple manner by mixing varioussolid components, in particular powder, granules or extrudates, havingvarious ingredients and/or different release behavior with one anotherin an overall loose mixture. Solid agents according to the inventionwhich consist of one or more phases may be prepared in the known manner,for example by spray-drying or granulation, adding the enzymes andpossible further thermosensitive ingredients such as, for example,bleaches, separately later, where appropriate. To prepare agentsaccording to the invention having an increased bulk density, inparticular in the range from 650 g/l to 950 g/l, preference is given toa method which has an extrusion step and has been disclosed in Europeanpatent EP 0 486 592. European patent EP 0 642 576 describes anotherpreferred preparation with the aid of a granulation process.

Proteins may be used in dried, granulated, encapsulated, or encapsulatedand additionally dried form, for example, for solid agents. They may beadded separately, i.e. as a separate phase, or together with othercomponents in the same phase, with or without compaction. Ifmicroencapsulated enzymes are to be processed in solid form, it ispossible to remove the water from the aqueous solutions resulting fromthe workup by using methods known in the prior art, such asspray-drying, removing by centrifugation or resolubilizing. Theparticles obtained in this way are usually between 50 and 200 μm insize.

The encapsulated form is a way of protecting the enzymes from othercomponents such as, for example, bleaches, or of making a controlledrelease possible. Depending on their size, the capsules are divided intomilli-, micro- and nanocapsules, microcapsules being particularlypreferred for enzymes. Another possible encapsulation method is toencapsulate the enzymes suitable for use in detergents or cleansers,starting from a mixture of the enzyme solution with a solution orsuspension of starch or a starch derivative, into starch or said starchderivative.

It is also possible for least two phases to be associated with oneanother. Thus, compression or compacting to give tablets is anotherpossibility of providing a solid agent according to the invention. Suchtablets may have one or more phases. Consequently, this presentationform also offers the possibility of providing a two-phase solid agentaccording to the invention. To produce agents according to the inventionin tablet form, which may have one or more phases, may have one or morecolors and/or consist of one or more layers, preference is given tomixing all of the components—per one layer, where appropriate—with oneanother in a mixer and compressing said mixture by means of conventionaltabletting presses, for example eccentric presses or rotary presses, atpressing forces in the range of from about 50 to 100 kN/cm2, preferablyat from 60 to 70 kN/cm2. Particularly in the case of multilayer tablets,it may be of advantage if at least one layer is compressed beforehand.This is preferably accomplished at pressing forces of between 5 and 20kN/cm2, in particular at from 10 to 15 kN/cm2. A tablet produced in thisway preferably has a weight of from 10 g to 50 g, in particular from 15g to 40 g. The three dimensional form of the tablets is arbitrary andmay circular, oval or angular, with intermediate forms also beingpossible.

At least one of the phases in multi-phase agents, that contains anamylase-sensitive material, in particular starch, or is at leastpartially surrounded or coated by said material, is particularlyadvantageous. Said phase is mechanically stabilized and/or protectedfrom influences from the outside in this way and is, at the same time,attacked via an amylase active in the wash liquor, so as to facilitaterelease of the ingredients.

Agents according to the invention that are likewise preferred arecharacterized in that they are overall in a liquid, gel-like orpaste-like form. The proteins contained therein, preferably a protein ofthe invention, are added to such agents, preferably starting fromprotein isolation and preparation carried out according to the priorart, in a concentrated aqueous or nonaqueous solution, for example inliquid form, for example as solution, suspension or emulsion, but alsoin gel form or encapsulated or as dried powder. Such detergents orcleansers according to the invention in the form of solutions incustomary solvents are usually prepared by simply mixing the ingredientswhich can be introduced as solids or as a solution into an automatedmixer.

One embodiment of the present invention are those agents in liquid, gelor paste form, to which a protein essential to the invention and/or anyof the other contained proteins and/or any of the other containedingredients has been added in encapsulated form, preferably in the formof microcapsules. Among these, particular preference is given to thosewith capsules made of amylase-sensitive material. Such a combined use ofamylase-sensitive materials and the amylolytic enzyme essential to theinvention in a detergent or cleanser may exhibit synergistic effects,for example such that the starch-cleaving enzyme assists cleavage of themicrocapsules and thus controls the process of releasing theencapsulated ingredients so that the latter are released not duringstorage and/or not at the beginning of the purification process but onlyat a particular time. This mechanism may be the basis of complexdetergent and cleanser systems with a large variety of ingredients andcapsule types, which are particularly preferred embodiments of thepresent invention.

A comparable effect arises when the ingredients of the detergent orcleanser are distributed over at least two different phases, for exampletwo or more solid phases, connected to one another, of a tablet-likedetergent or cleanser, or different granules within the same pulverulentagent. Two- or multiphase cleaners are state of the art for applicationboth in machine dishwashers and in detergents. The activity of anamylolytic enzyme in a previously activated phase is a precondition foractivation of a later phase, if the latter is surrounded by anamylase-sensitive envelope or coating or if the amylase-sensitivematerial is an integral component of the solid phase, which, whenpartially or completely hydrolyzed, leads to disintegration of the phasein question. The use of the enzyme essential to the invention for thispurpose is thus a preferred embodiment of the present invention.

The ingredients of detergents and cleansers are suitably able to supporteach other's performance. The application WO 99/63035, for example,discloses the synergistic use of amylase and color transfer inhibitorsin order to increase cleaning performance. It has also been disclosed,for example in the application WO 98/45396, that polymers which may beused simultaneously as cobuilders, such as, for example, alkylpolyglycosides, can stabilize and increase the activity and stability ofenzymes present. Preference is therefore given to an α-amylase variantaccording to the invention being modified, in particular stabilized,and/or its contribution to the washing or cleaning performance of theagent being increased by any of the other components mentioned above.Appropriately adjusted formulations for agents according to theinvention are thus particularly preferred embodiments of the presentinvention.

Within appropriate agents, α-amylase variants according to the inventionmay serve to activate their own or other phases, if they are providedalone or together with at least one other cleaning-active or cleaningaction-supporting substance in a detergent or cleanser consisting ofmore than one phase. Accordingly, they may also serve to releaseingredients from capsules, if they or another active substance areprovided in encapsulated form in a detergent or cleanser.

The invention also relates to methods of cleaning textiles or hardsurfaces, which are characterized in that an above-described α-amylasevariant according to the invention becomes active in at least one of theprocess steps.

This is because this embodiment implements the invention in that theimproved enzymic properties according to the invention, in particularthe increased stability to multimerization, are beneficial in principleto any cleaning process, for example in that less enzyme is lost due toaggregation, even during application. Each cleaning process is enrichedby the activity in question, if the latter is added in at least oneprocess step. Processes of this kind are carried out, for example, usingmachines such as familiar domestic dishwashers or domestic washingmachines. Preference is accordingly given to preferred processesaccording to the above statements.

Further preference is given to those methods which are characterized inthat the α-amylase variant is employed by way of an above-describedagent according to the invention.

Particular preference is given to any method characterized in that theα-amylase is employed in the step in question in an amount of from 0.01mg to 400 mg per corresponding step, preferably from 0.02 mg to 300 mg,particularly preferably from 0.03 mg to 100 mg.

Advantageously, this results in concentrations of from 0.0005 to 20 mgper I, preferably 0.005 to 10 mg per I, particularly preferably 0.005 to8 mg of the amylolytic protein per I of wash liquor. The proteinconcentration may be determined with the aid of known methods, forexample the abovementioned BCA or biuret methods.

According to the above specification, the present invention is alsoimplemented by the use of α-amylase variants of the invention becausehere too, the advantageous properties of the enzymes in question areeffective. This applies in particular to cleaning purposes.

A separate subject matter of the invention is therefore the use of anyof the above-described α-amylase variants according to the invention forcleaning textiles or hard surfaces.

It is possible here to use said variant as the sole active component,preferentially together with at least other cleaning-active or cleaningaction-supporting substance.

Preference is therefore given to such a use that is characterized inthat the α-amylase variant is employed by way of an above-describedagent according to the invention.

Advantageously, such a use is characterized in that from 0.01 mg to 400mg of the α-amylase variant, preferably from 0.02 mg to 300 mg,particularly preferably from 0.03 mg to 100 mg, are employed perapplication, preferably per application in a dishwasher or a washingmachine.

This is because this produces advantageously the concentrations listedabove in the wash liquor. This metering may be carried out by themanufacturer of said agent or by the end user, depending on the cleaningproblem.

Another embodiment is the use of an α-amylase variant according to theinvention for the treatment of raw materials or intermediate products intextile manufacture, in particular for desizing cotton.

Raw materials and intermediates in the manufacture of textiles, forexample those based on cotton, are provided with starch during theirproduction and further processing, in order to improve processing. Thismethod which is applied to yarns, to intermediates and to textiles iscalled sizing. Amylolytic proteins according to the invention aresuitable for removing the starch-containing protective layer (desizing),in particular because they are stabilized to multimerization during theaction in a liquid medium.

The following examples further illustrate the present invention.

EXAMPLES

All molecular-biological steps are carried out following standardmethods as described, for example, in the manual by Fritsch, Sambrookand Maniatis “Molecular cloning: a laboratory manual”, Cold SpringHarbour Laboratory Press, New York, 1989, or comparable specialistliterature. Enzymes, kits and apparatus are used according to theinstructions by the respective manufacturers.

Example 1 Culturing of Bacillus sp. A 7-7 (DSM 12368)

The microorganism Bacillus sp. A 7-7 has been deposited under thedeposit number DSM 12368 with the Deutsche Sammlung von Mikroorganismenand Zellkulturen gmbH, Mascheroder Weg 1 b, 38124 Braunschweig, Germany(http://www.dsmz.de). It is described in the application WO 02/10356 A2.The DNA and amino acid sequences of the α-amylase produced by this(deposited) species differ from the sequences depicted in the sequencelisting of WO 02/10356 A2 in the following two positions: thecorresponding DNA has, in nucleic acid positions 805-807 according toSEQ ID NO. 1 of the present application, the triplet gat which codes forthe amino acid D (in amino acid position 236), and, in positions1156-1158, the triplet tat which codes for the amino acid Y (in position353). (SEQ ID NO. 1 and 2 of WO 02/10356 A2 indicate the triplets ggtfor G and tgt for C, respectively, at the corresponding positions.)

Using well-known methods of point mutagenesis, for example with the aidof the QuickChange kit from Stratagene®, La Jolla, USA (see below), andon the basis of primers derived from SEQ ID NO. 1, it is possible toconvert this α-amylase to a different one.

WO 02/10356 A2 describes culturing of Bacillus sp. A 7-7 (DSM 12368). Asuitable medium is YPSS medium containing 15 g/l soluble starch, 4 g/lyeast extract, 1 g/l K₂HPO₄ and 0.5 g/l mgSO₄×7H₂O, with the pH beingadjusted to 10.3 with 20% strength sodium carbonate solution afterautoclaving. From this the α-amylase in question can be obtained, aslikewise illustrated in WO 02/10356 A2. Thus it is possible for saidα-amylase and the variants derived therefrom to be prepared bywell-known methods, at least on the laboratory scale.

Example 2 Homology Modeling and Selection of the Amino Acids ReplaceableAccording to the Invention Homology Modeling

Homology modeling for Bacillus sp. A 7-7 (DSM 12368) α-amylase wascarried out via the RSCB protein database (accessible viaMax-Delbrück-Zentrum in Berlin, Germany), as illustrated in thedescription. In this connection, the search using the protein sequencedetected the following structures: B. licheniformis α-amylase (RCSB-PDBdatabase entry: 1 BLI), a chimeric α-amylase of those of B.amyloliquefaciens and B. licheniformis, in its native structure (1E3X,1E43), an acarbose complex of the same chimeric α-amylase (1E3Z), aTris/maltotriose complex of the same chimeric α-amylase (1E40) and akinetically stabilized variant of B. licheniformis α-amylase (10B0).

By superimposing these structures in the SwissPDB viewer, a leaderstructure was generated onto which the protein sequence of ALBA was thenmodeled. The orientation of the side chains in the ALBA structure wasthen corrected and energy minimization was carried out. The individualsteps can be found in the user manual mentioned and were carried outaccording to the standard settings of the program.

Overall, the following 407 amino acids are located on the surface of themolecule which comprises 484 amino acids (they are defined as such byway of an accessibility of at least 1; regarding accessibility: seedescription and subsequent section):

T5, N6, G7, T8, M9, Q11, Y12, E14, W15, Y16, L17, P18, N19, D20, G21,N22, H23, W24, N25, R26, R28, S29, D30, A31, S32, N33, K35, D36, K37,G38, 139, T40, A41, W43, P46, A47, W48, K49, G50, A51, S52, Q53, N54,D55, V56, G57, Y58, G59, A60, Y61, D62, L63, Y64, L66, G67, E68, F69,N70, Q71, K72, G73, T74, V75, R76, T77, K78, Y79, G80, T81, R82, N83,Q84, L85, Q86, A87, V89, T90, A91, K93, S94, N95, G96, Q98, V99, Y100,V103, M105, N106, H107, K108, G110, A111, D112, A113, T114, E115, W116,V117, R118, V120, E121, V122, N123, P124, S125, N126, R127, N128, Q129,E130, V131, S132, G133, D134, Y135, T136, 1137, E138, W140, K142, F143,D144, F145, P146, G147, R148, G149, N150, T151, H152, S153, N154, F155,K156, W157, R158, W159, Y160, H161, D166, W167, D168, Q169, S170, R171,Q172, L173, Q174, N175, R176, I177, Y178, K179, R181, G182, D183, G184,K185, G186, W187, W189, E190, V191, D192, T193, E194, N195, G196, N197,Y198, D199, Y200, L201, M202, Y203, I206, D207, M208, D209, H210, P211,E212, V214, N215, E216, L217, R218, N219, V222, W223, T225, N226, T227,L228, G229, L230, D231, F233, R234, 1235, G236, A237, K239, H240, 1241,K242, Y243, S244, F245, T246, R247, D248, W249, L250, T251, H252, V253,R254, N255, T256, T257, G258, K259, N260, M261, F262, A263, E266, F267,W268, K269, N270, D271, I272, G273, A274, I275, E276, N277, S280, K281,N283, W284, N285, H286, S287, V288, F289, P292, L293, Y295, N296, L297,Y298, N299, S301, R302, S303, G304, G305, N306, Y307, D308, M309, R310,Q311, 1312, F313, N314, G315, V318, Q319, R320, H321, P322, T323, H324,T327, F328, V329, D330, N331, H332, D333, Q335, P336, E337, E338, A339,L340, E341, S342, F343, E345, E346, W347, F348, K349, P350, L351, C353,L355, T356, L357, R359, D360, Q361, G362, Y363, S365, V366, F367, Y368,D370, Y371, Y372, F373, 1374, P375, T376, H377, F378, P380, A381, M382,K383, S384, K385, 1386, D387, P388, L390, E391, R393, Q394, K395, Y396,Y398, G399, K400, Q401, N402, D403, Y404, L405, D406, H407, H408, N409,M410, R415, E416, G417, N418, T419, A420, H421, P422, N423, S424, M430,D432, G433, P434, G435, G436, N437, K438, W439, Y441, G443, R444, N445,K446, A447, G448, Q449, V450, W451, R452, D453, 1454, T455, G456, N457,R458, S459, G460, T461, V462, T463, I464, N465, A466, D467, W469, N471,S473, V474, N475, G476, G477, S478, V479, V483, N484, N485

Calculation of the Surface Amino Acid Residues Contributing to theElectrostatic Potential of the Whole Molecule

Determination of the three-dimensional structure is followed by acalculation of the particular contributions of the amino acids on thesurface to the electrostatic potential of the whole molecule. Thiscalculation too was carried out as specified in the description, usingthe corresponding function of the mentioned SwissPDB viewer withstandard parameters. Such contributions are made both by neutral andnegatively charged amino acid residues and by those which themselves areneutral but cover a charge further inside the molecule. This istherefore a projection of the charges onto the molecular surface.

The result thereof is depicted in FIG. 1: the three-dimensionalrepresentation of the Conolly surface of Bacillus sp. A 7-7 (DSM 12368)α-amylase is visible there, with the charge and polarity distributionbeing highlighted by color (white, gray and black). Overall, thefollowing 118 amino acid residues of the Bacillus sp. A 7-7 (DSM 12368)α-amylase molecule make a positive or neutral contribution to theelectrostatic potential of the surface:

T5, N6, G7, T8, N19, G21, N22, H23, N25, R26, R28, S29, A31, S32, N33,K35, K37, G38, K49, Q53, L66, K72, T74, V75, R76, K78, T81, R82, N83,Q84, L85, Q86, A87, V89, T90, A91, K93, S94, N95, G96, Q98, K108, R118,T136, K142, G149, N150, T151, H152, N154, K156, R158, Y160, H161, R171,Q172, R176, R181, R218, T227, L228, G229, K242, R247, T251, R254, K259,N260, K281, N283, R302, R310, R320, T323, R359, Y368, Y372, T376, K383,K385, R393, Q394, K395, Y398, G399, K400, Y404, M410, R415, G417, N418,T419, A420, H421, P422, G435, G436, K438, W439, R444, N445, K446, Q449,V450, R452, R458, S459, G460, T461, V462, T463, N465, A466, N471, S473,N475, G476, N484.

Calculation of Solvent Accessibility

Based on these results, the solvent accessibility of the above-describedamino acid residues which are located on the surface and make a positiveor neutral contribution to the charge or polarity of the whole moleculewere then calculated. To this end, the above-mentioned SwissPdb viewerwas used again, preserving the standard parameters of the program. As aresult, the following amino acid residues were determined the solventaccessibility values being indicated in each case in % in brackets afterthe positions listed:

T5 (39), N6 (12), G7 (13), N19 (28), N22 (28), N25 (16), R26 (27), R28(39), S29 (38), S32 (28), N33 (28), K35 (37), K37 (18), Q53 (12), K72(27), V75 (30), R76 (24), T81 (16), R82 (22), N83 (44), Q84 (24), Q86(18), A87 (18), T90 (30), A91 (11), K93 (32), S94 (50), N95 (19), G96(25), Q98 (29), R118 (41), T136 (22), K142 (30), G149 (14), N150 (39),T151 (22), H152 (27), N154 (41), K156 (30), R158 (33), Y160 (20), R171(32), Q172 (53), R176 (41), R181 (34), R218 (18), T227 (19), G229 (14),K242 (15), R247 (20), T251 (23), R254 (15), K259 (26), N260 (49), K281(33), N283 (40), R302 (50), R310 (31), R320 (52), T323 (49), R359 (13),Y368 (12), Y372 (37), T376 (56), K383 (19), K385 (37), Q394 (20), K395(38), G399 (16), K400 (44), Y404 (11), G417 (11), N418 (25), T419 (59),A420 (37), H421 (16), P422 (46), G435 (25), G436 (17), W439 (47), R444(49), N445 (41), Q449 (31), V450 (24), R452 (33), R458 (24), S459 (52),G460 (32), T461 (35), T463 (40), N465 (22), A466 (37), N471 (20), S473(10), N475 (25), G476 (31), N484 (12).

These are thus the 97 amino acid residues of the 484 amino acids intotal of the Bacillus sp. A 7-7 (DSM 12368) α-amylase molecule, whichmake a positive or neutral contribution to the electrostatic potentialof the surface and additionally have an accessibility of at least 10%.

The remaining 21 surface amino acids which had been determinedpreviously as those making a neutral or positive contribution but havingan accessibility of less than 10% are the following, with thecorresponding value again being indicated in brackets:

T8 (2), G21 (4), H23 (2), A31 (2), G38 (6), K49 (2), L66 (2), T74 (6),K78 (6), L85 (2), V89 (1), K108 (9), H161 (1), L228 (1), R393 (5), Y398(4), M410 (3), R415 (6), K438 (7), K446 (5), V462 (5).

Grouping of the Neutral or Positively Charged or Polarized Amino AcidResidues Particularly Accessible to the Solvent

The 97 identified residues having an accessibility of at least 10% maybe assigned to various groups according to their location on the surfaceof said α-amylase, with those of groups A and B representing in eachcase contiguous regions of neutral or positive polarity or charge. groupA below comprises the 63 amino acid residues among them, which form acontiguous surface with neutral or positive electrostatic potential(with the previously determined accessibility again in brackets):

-   (A) T5 (39), N6 (12), G7 (13), N19 (28), N22 (28), N25 (16), R26    (27), R28 (39), S29 (38), S32 (28), N33 (28), K35 (37), K37 (18),    Q53 (12), K72 (27), V75 (30), R76 (24), T81 (16), R82 (22), N83    (44), Q84 (24), Q86 (18), A87 (18), T90 (30), A91 (11), K93 (32),    S94 (50), N95 (19), G96 (25), Q98 (29), R118 (41), T136 (22), K142    (30), G149 (14), N150 (39), T151 (22), H152 (27), N154 (41), K156    (30), R158 (33), Y160 (20), R171 (32), Q172 (53), R181 (34), T227    (19), G229 (14), R247 (20), T251 (23), R254 (15), K259 (26), N260    (49), K281 (33), N283 (40), Q394 (20), K395 (38), G399 (16), K400    (44), G417 (11), N418 (25), T419 (59), A420 (37), H421 (16), P422    (46).

Group B below comprises the 20 amino acid residues forming a secondcontiguous surface with a neutral or positive electrostatic potential:

-   (B) G435 (25), G436 (17), W439 (47), R444 (49), N445 (41), Q449    (31), V450 (24), R452 (33), R458 (24), S459 (52), G460 (32), T461    (35), T463 (40), N465 (22), A466 (37), N471 (20), S473 (10), N475    (25), G476 (31), N484 (12).

Group C below comprises the remaining 14 amino acid residues whichcannot be assigned to any of said two larger areas but occur isolated:

-   (C) R176 (41), R218 (18), K242 (15), R302 (50), R310 (31), R320    (52), T323 (49), R359 (13), Y368 (12), Y372 (37), T376 (56), K383    (19), K385 (37), Y404 (11).

The amino acids belonging to groups A and B, in particular, can beassumed to contribute with their contributions to a contiguous surfacecharge to the observed tendency of charge- and/or polarity-mediated di-and/or multimerization and therefore to aggregation.

Example 3 Site-Specific Mutagenesis

For Bacillus sp. A 7-7 (DSM 12368) α-amylase, the positions determinedin the previous example serve as starting points for point mutations viasite-directed mutagenesis, i.e. for introducing a different amino acidto the position(s) in question. Said mutagenesis is carried out, forexample, with the aid of the QuikChange kit (Stratagene, cat. NO.200518) according to the corresponding protocol. The primers may bedesigned on the basis of the DNA and amino acid sequences indicated inSEQ ID NO. 1, the particular codon being altered according to the aminoacid to be introduced. This involves the possible amino acidsubstitutions below for generating a less neutral or positive polarityor charge, i.e. for introducing a rather negative polarity or charge:

Starting amino acid to give Arg (R) K, Y, C, H, G, A, V, L, I, M, F, W,P, S, T, N, Q, E or D Lys (K) Y, C, H, G, A, V, L, I, M, F, W, P, S, T,N, Q, E or D Tyr (Y) C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or DCys (C) H, G, A, V, L, I, M, F, W, P, S, T, N, Q, E or D His (H) G, A,V, L, I, M, F, W, P, S, T, N, Q, E or D Gly (G) A, V, L, I, M, F, W, P,S, T, N, Q, E or D Ala (A) V, L, I, M, F, W, P, S, T, N, Q, E or D Val(V) L, I, M, F, W, P, S, T, N, Q, E or D Leu (L) I, M, F, W, P, S, T, N,Q, E or D Ile (I) M, F, W, P, S, T, N, Q, E or D Met (M) F, W, P, S, T,N, Q, E or D Phe (F) W, P, S, T, N, Q, E or D Trp (W) P, S, T, N, Q, Eor D Pro (P) S, T, N, Q, E or D Ser (S) T, N, Q, E or D Thr (T) N, Q, Eor D Asn (N) Q, E or D Gln (Q) E or D Glu (E) D

To this end, the codon in the gene sequence of the α-amylase in questionis thus replaced with a codon of the amino acid to be introduced.According to this principle, an expression vector containing theα-amylase sequence is suitably mutagenized accordingly and transformedinto an expression strain in the present example into B. subtilis, bywell-known methods.

Example 4 Production and Purification of Amylase Mutants

Amylase-positive B. subtilis strains are grown in the YPSS mediummentioned in Example 1. This procedure and purification of the enzymeproduced by these strains are carried out according to the descriptionin WO 02/10356 A2. The latter also reveals determination of theamylolytic activity of the purified enzyme according to the “DNSmethod”. The activity determinable in this way serves hereinbelow asparameter for the stability of the enzyme under in each case differentconditions.

Determination of Aggregate Formation

The formation of multimers and precipitate is detected by way of theturbidity of an amylase-containing solution having an amylase content ofat least 5 mg/ml spectrometrically at a wavelength of 600 nm. Mutantsaccording to the invention exhibit a reduced tendency to formprecipitate after 16 hours of incubation at a concentration of at least5 mg/ml protein in buffer or culture medium at 25° C., which tendency isexpressed as a reduced increase in absorption at 600 nm.

ABBREVIATIONS

A 7-7: α-amylase of Bacillus sp. A 7-7 (DSM 12368; SEQ ID NO. 2)

S707: α-amylase of Bacillus sp. #707 (SEQ ID NO. 3)

LAMY: α-amylase of Bacillus sp. KSM-AP1378 (SEQ ID NO. 4)

BAA: α-amylase of B. amyloliquefaciens (SEQ ID NO. 5)

BLA: α-amylase of B. licheniformis (SEQ ID NO. 6)

BStA: α-amylase of B. stearothermophilus (SEQ ID NO. 7)

MK716: α-amylase of Bacillus sp. MK716 (SEQ ID NO. 8)

TS-23: α-amylase of Bacillus sp. TS-23 (SEQ ID NO. 9)

K38: α-amylase of Bacillus sp. KSM-K38 (SEQ ID NO. 10)

1-47. (canceled)
 48. A cell comprising a nucleic acid coding for analpha-amylase variant of the alpha-amylase of Bacillus sp. A 7-7 (DSM12368) according to positions +1 to 484 of SEQ ID NO: 2 and havingalpha-amylase activity, the variant having a plurality of amino acidsubstitutions with respect to the alpha-amylase starting molecule,wherein the alpha-amylase variant differs from the alpha-amylasestarting molecule by substitution of a plurality of predeterminedstarting amino acid residues at the surface of the molecule making aneutral or positively polar or charged contribution to the electrostaticpotential of said molecule with more negatively polar or negativelycharged substituted amino acid residues, thereby providing increasedstability to aggregate formation, wherein the amino acid substitutionsare chosen from the following substitutions: Starting amino acidSubstituted amino acid Arg (R) K, Y, C, H, G, A, V, L, I, M, F, W, P, S,T, N, Q, E or D Lys (K) Y, C, H, G, A, V, L, I, M, F, W, P, S, T, N, Q,E or D Trp (W) P, S, T, N, Q, E or D Pro (P) S, T, N, Q, E or D Ser (S)T, N, Q, E or D Thr (T) N, Q, E or D Asn (N) Q, E or D Gln (Q) E or D

wherein said starting amino acid residues are selected from thefollowing positions: 83, 94, 118, 154, 172, 176, 260, 283, 302, 320,323, 376, 400, 419, 422, 439, 444, 445, 459, and 463, in each caseindicated in the numbering of the mature protein according to SEQ ID NO.2, wherein the amino acid substitutions are limited to two or more ofthe listed positions, and wherein the number of substitutions is up to20.
 49. The cell of claim 48, wherein said nucleic acid is part of avector.
 50. The cell of claim 48, wherein the cell is a bacterium thatsecretes the alpha-amylase variant into the surrounding medium.
 51. Thecell of claim 48, wherein the cell is a Gram-negative bacterium selectedfrom the group consisting of the species Escherichia coli, the genusKlebsiella, the genus Pseudomonas, and the genus Xanthomonas.
 52. Thecell of claim 51, wherein the cell is selected from the group consistingof the strains Escherichia coli BL21 (DE3), E. coli RV308, E. coliDH5alpha, E. coli JM109, E. coli XL-1, and Klebsiella planticola (Rf).53. The cell of claim 48, wherein the cell is a Gram-positive bacteriumselected from the group consisting of the genera Bacillus,Staphylococcus, and Corynebacterium.
 54. The cell according to claim 53,wherein the cell is selected from the group consisting of the speciesBacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B.globigii, B. alcalophilus, Staphylococcus carnosus, and Corynebacteriumglutamicum.